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Abstract:

The present invention provides compositions and methods of use of
humanized, chimeric or human Class I anti-CEA antibodies or fragments
thereof, preferably comprising the light chain variable region CDR
sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT
(SEQ ID NO:3); and the heavy chain variable region CDR sequences DYYMS
(SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID
NO:6). The Class I anti-CEA antibodies or fragments are useful for
treating diseases, such as cancer, wherein the diseased cells express
CEACAM5 and/or CEACAM6 antigens. The Class I anti-CEA antibodies or
fragments are also of use for interfering with specific processes, such
as metastasis, invasiveness and/or adhesion of cancer cells, or for
enhancing sensitivity of cancer cells to cytotoxic agents and have
favorable effects on the survival of subjects with cancer.

2. The method of claim 1, wherein the Class I anti-CEA antibody or
fragment thereof is a naked antibody or fragment thereof.

3. The method of claim 2, further comprising administering at least one
therapeutic agent to the subject.

4. The method of claim 3, wherein the therapeutic agent is selected from
the group consisting of a naked antibody, an antibody fragment, a
cytotoxic agent, a drug, a radionuclide, boron atoms, an immunomodulator,
a photoactive therapeutic agent, an immunoconjugate, an oligonucleotide
and a hormone.

5. The method of claim 1, wherein the Class I anti-CEA antibody or
fragment thereof is conjugated to at least one therapeutic agent.

6. The method of claim 5, wherein the therapeutic agent is selected from
the group consisting of an antibody, an antibody fragment, a cytotoxic
agent, a drug, a radionuclide, boron atoms, an immunomodulator, a
photoactive therapeutic agent, an immunoconjugate, an oligonucleotide and
a hormone.

7. The method of claim 6, wherein the cytotoxic agent is a drug or toxin.

8. The method of claim 7, wherein the drug has a pharmaceutical property
selected from the group consisting of antimitotic, alkylating,
antimetabolite, antiangiogenic, apoptotic, alkaloid, COX-2, and
antibiotic agents.

16. The method of claim 14, wherein the chemokine is selected from the
group consisting of RANTES, MCAF, MIP1-alpha, MIP1-beta and IT-10.

17. The method of claim 1, wherein the chimeric, humanized or human Class
I anti-CEA antibody or fragment is part of a bispecific antibody further
comprising a second antibody or fragment thereof that binds to a
targetable construct and the method further comprises administering a
targetable construct to the subject, wherein the targetable construct is
conjugated to at least one therapeutic agent.

18. The method of claim 17, wherein the therapeutic agent is selected
from the group consisting of an antibody, an antibody fragment, a
cytotoxic agent, a drug, a radionuclide, boron atoms, an immunomodulator,
a photoactive therapeutic agent, an immunoconjugate, an oligonucleotide
and a hormone.

19. The method of claim 1 wherein the chimeric, humanized or human Class
I anti-CEA antibody or fragment is a fusion protein.

28. The method of claim 1, wherein the antibody fragment is selected from
the group consisting of F(ab')2, Fab', Fab, Fv and scFv.

29. The method of claim 1, wherein the disease is an infection.

30. The method of claim 1, wherein the disease is an inflammation.

31. The method of claim 1, wherein the disease is sepsis.

Description:

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.
No. 12/846,062, filed Jul. 29, 2010. The present application claims the
benefit under 35 U.S.C. 119(e) of Provisional U.S. Patent Application
Ser. No. 61/242,872, filed Sep. 16, 2009, the entire text of which is
incorporated herein by reference.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been
submitted via EFS-Web and is hereby incorporated by reference in its
entirety. Said ASCII copy, created on Jul. 9, 2010, is named
IMM322US.txt, and is 17,798 bytes in size.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The invention relates to compositions for and methods of treating
cancers that express CEACAM 5 (carcinoembryonic antigen, "CEA") and/or
CEACAM6 (NCA-90), such as medullary thyroid cancer (MTC), colorectal
cancers, hepatocellular carcinoma, gastric cancer, lung cancer,
head-and-neck cancers, bladder cancer, prostate cancer, breast cancer,
pancreatic cancer, uterine cancer, ovarian cancer, hematopoietic cancers,
leukemia and other cancers in which CEACAM5 and/or CEACAM6 are expressed.
The methods comprise administering a Class I anti-CEA antibody or
fragment that targets both CEACAM5 and CEACAM6, preferably in combination
with at least one other therapeutic agent, such as another antibody, a
chemotherapeutic agent, a radioactive agent, an antisense
oligonucleotide, an immunomodulator, an immunoconjugate or a combination
thereof. The Class I anti-CEA MAb may be administered prior to, with or
after administering the therapeutic agent. In preferred embodiments the
Class I anti-CEA antibody is a chimeric, humanized or human monoclonal
antibody (MAb) comprising the light chain variable region CDR sequences
SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ ID
NO:3); and heavy chain variable region CDR sequences DYYMS (SEQ ID NO:4);
FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6). More
preferably, the chimeric, humanized or human Class I anti-CEA MAb retains
the binding affinity characteristics and specificities of a parental
murine Class I anti-CEA MAb, but possesses more of the antigenic and
effector properties of a human antibody.

[0005] 2. Related Art

[0006] CEA (CEACAM5) is an oncofetal antigen commonly expressed in a
number of epithelial cancers, most commonly those arising in the colon
but also in the breast, lung, pancreas, thyroid (medullary type) and
ovary (Goldenberg et al., J. Natl. Cancer Inst. 57: 11-22, 1976; Shively,
et al., Crit. Rev. Oncol. Hematol. 2:355-399, 1985). The human CEA gene
family is composed of 7 known genes belonging to the CEACAM subgroup.
These subgroup members are mainly associated with the cell membrane and
show a complex expression pattern in normal and cancerous tissues. The
CEACAM5 gene, also known as CD66e, codes for the CEA protein (Beauchemin
et al., Exp Cell Res 252:243, 1999). CEACAM5 was first described in 1965
as a gastrointestinal oncofetal antigen (Gold et al., J Exp Med
122:467-481, 1965), but is now known to be overexpressed in a majority of
carcinomas, including those of the gastrointestinal tract, the
respiratory and genitourinary systems, and breast cancer (Goldenberg et
al., J Natl Cancer Inst. 57:11-22, 1976; Shively and Beatty, Crit. Rev
Oncol Hematol 2:355-99, 1985).

[0007] CEACAM6 (also called CD66c or NCA-90) is a non-specific
cross-reacting glycoprotein antigen that shares some, but not all,
antigenic determinants with CEACAM5 (Kuroki et al., Biochem Biophys Res
Comm 182:501-06, 1992). CEACAM6 is expressed on granulocytes and
epithelia from various organs, and has a broader expression zone in
proliferating cells of hyperplastic colonic polyps and adenomas, compared
with normal mucosa, as well as by many human cancers (Scholzel et al., Am
J Pathol 157:1051-52, 2000; Kuroki et al., Anticancer Res 19:5599-5606,
1999). Relatively high serum levels of CEACAM6 are found in patients with
lung, pancreatic, breast, colorectal, and hepatocellular carcinomas. The
amount of CEACAM6 does not correlate with the amount of CEACAM5 expressed
(Kuroki et al., Anticancer Res 19:5599-5606, 1999).

[0009] Anti-CEA antibodies are classified into different categories,
depending on their cross-reactivity with antigens other than CEA.
Anti-CEA antibody classification was described by Primus and Goldenberg,
U.S. Pat. No. 4,818,709 (incorporated herein by reference from Col. 3,
line 5 through Col. 26, line 49 of U.S. Pat. No. 4,818,709). The
classification of anti-CEA antibodies is determined by their binding to
CEA, meconium antigen (MA) and nonspecific crossreacting antigen (NCA).
Class I anti-CEA antibodies bind to all three antigens. Class II
antibodies bind to MA and CEA, but not to NCA. Class III antibodies bind
only to CEA (U.S. Pat. No. 4,818,709). Examples of each class of anti-CEA
antibody are known, such as MN-3, MN-15 and NP-1 (Class I); MN-2, NP-2
and NP-3 (Class II); and MN-14 and NP-4 (Class III) (U.S. Pat. No.
4,818,709; Blumenthal et al. BMC Cancer 7:2 (2007)).

[0010] The epitopic binding sites of various anti-CEA antibodies have also
been identified. The MN-15 antibody binds to the A1B1 domain of CEA, the
MN-3 antibody binds to the N-terminal domain of CEA and the MN-14
antibody binds to the A3B3 (CD66e) domain of CEA (Blumenthal et al. BMC
Cancer 7:2 (2007)). There is no direct correlation between epitopic
binding site and class of anti-CEA antibody. For example, MN-3 and MN-15
are both Class I anti-CEA antibodies, reactive with NCA, MA and CEA, but
bind respectively to the N-terminal and A1B1 domains of CEA. Primus and
Goldenberg (U.S. Pat. No. 4,818,709) reported a complicated pattern of
cross-blocking activity between the different anti-CEA antibodies, with
NP-1 (Class I) and NP-2 (Class II) cross-blocking binding to CEA of each
other, but neither blocking binding of NP-3 (Class II). However, by
definition Class I anti-CEA antibodies bind to both CEACAM5 and CEACAM6,
while Class III anti-CEA antibodies bind only to CEACAM5.

[0011] Anti-CEA antibodies have been suggested for therapeutic treatment
of a variety of cancers. For example, medullary thyroid cancer (MTC)
confined to the thyroid gland is generally treated by total thyroidectomy
and central lymph node dissection. However, disease recurs in
approximately 50% of these patients. In addition, the prognosis of
patients with unresectable disease or distant metastases is poor, less
than 30% survive 10 years (Rossi et al., Amer. J. Surgery, 139:554
(1980); Samaan et al., J. Clin. Endocrinol. Metab., 67:801 (1988);
Schroder et al., Cancer, 61:806 (1988)). These patients are left with few
therapeutic choices (Principles and Practice of Oncology, DeVita, Hellman
and Rosenberg (eds.), New York: JB Lippincott Co., pp. 1333-1435 (1989);
Cancer et al., Current Problems Surgery, 22: 1 (1985)). The Class III
anti-CEA antibody MN-14 has been reported to be effective for therapy of
human medullary thyroid carcinoma in an animal xenograft model system,
when used in conjunction with pro-apoptotic agents such as DTIC, CPT-11
and 5-fluorouracil (U.S. patent application Ser. No. 10/680,734, the
Examples section of which is incorporated herein by reference). The Class
III anti-CEA antibody reportedly sensitized cancer cells to therapy with
chemotherapeutic agents and the combination of antibody and
chemotherapeutic agent was reported to have synergistic effects on tumors
compared with either antibody or chemotherapeutic agent alone (U.S. Ser.
No. 10/680,734). Anti-CEA antibodies of different classes (such as MN-3,
MN-14 and MN-15) have been proposed for use in treating a variety of
tumors.

[0012] There still exists a need to provide more effective methods of
treating CEA-expressing cancers. The present invention provides
compositions and methods for effective anti-cancer therapy utilizing
Class I anti-CEA MAbs, such as chimeric, humanized or human antibodies
comprising the light chain variable region CDR sequences SASSRVSYIH (SEQ
ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy
chain variable region CDR sequences DYYMS (SEQ ID NO:4);
FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6), which
are capable of binding to both CEACAM5 and CEACAM6. Preferably, the Class
I anti-CEA MAb is humanized, and used in combination with a therapeutic
agent, particularly a chemotherapeutic agent, to yield an effective
therapeutic treatment for CEACAM5- or CEACAM6-expressing cancers with
minimal toxicity. The separate administration of Class I antibody and
therapeutic agent provides enhanced results and the versatility and the
flexibility to tailor individual treatment methods.

[0014] In alternative embodiments, the chimeric, humanized or human Class
I anti-CEA MAb or antigen-binding fragment thereof may be one that blocks
or competes for binding to CEACAM5 and/or CEACAM6 with a monoclonal
antibody having light chain CDRs comprising CDR1 having an amino acid
sequence SASSRVSYIH (SEQ ID NO:1); CDR2 having an amino acid sequence
GTSTLAS (SEQ ID NO:2); and CDR3 having an amino acid sequence QQWSYNPPT
(SEQ ID NO:3); and heavy chain CDRs comprising CDR1 having an amino acid
sequence DYYMS (SEQ ID NO:4); CDR2 having an amino acid sequence
FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and CDR3 having an amino acid sequence
DMGIRWNFDV (SEQ ID NO:6). Such competitive binding or blocking studies
may be performed by any of a wide variety of known binding assays, as
exemplified in FIG. 1 and FIG. 4 and their corresponding Examples.

[0015] The skilled artisan will be aware, as discussed in more detail
below, that chimeric antibodies retain the framework region (FR)
sequences of a parent murine antibody, while humanized and human
antibodies will generally have human antibody FR sequences. Preferably,
the humanized Class I anti-CEA MAb comprises the heavy chain FR sequences
of the human KOL antibody and the light chain FR sequences of the human
REI antibody. However, where appropriate, certain murine FR amino acid
residues may be substituted for corresponding human FR amino acid
residues. In preferred embodiments, the substituted murine FR residues
may include one or more amino acid residues selected from heavy chain
amino acid residues 28, 29, 30, 48 and 49 of SEQ ID NO:10 and light chain
amino acid residues 21, 47 and 60 of SEQ ID NO:9. In more preferred
embodiments, the humanized Class I anti-CEA MAb comprises the variable
region sequences of SEQ ID NO:7 and SEQ ID NO:8, while the chimeric Class
I anti-CEA MAb comprises the variable region sequences of SEQ ID NO:9 and
SEQ ID NO:10.

[0016] In various embodiments, the Class I anti-CEA antibody will bind to
CEA, MA and NCA and will also bind to human granulocytes. The Class I
anti-CEA MAb binds to both CEACAM5 and CEACAM6. Where the Class I
anti-CEA MAb or fragment thereof is chimeric, humanized, or human, the
antibody will preferably retain the binding specificity of a parental
murine Class I anti-CEA MAb.

[0017] In certain embodiments the Class I anti-CEA MAb may be conjugated
to at least one therapeutic agent and/or diagnostic agent to form an
immunoconjugate. Such immunoconjugates are of use for delivering
therapeutic and/or diagnostic agents to a CEACAM5- or CEACAM6-expressing
cancer cell and/or for therapy or diagnosis of cancer. In alternative
embodiments, the Class I anti-CEA MAb may be administered as a "naked"
(unconjugated) antibody. Either naked antibodies or immunoconjugates may
be administered before, simultaneously with or after another therapeutic
anti-cancer agent.

[0018] Other embodiments concern methods of diagnosing or treating cancer,
comprising administering a Class I anti-CEA antibody or fragment thereof
to a subject. For diagnostic purposes, the antibody may be conjugated to
at least one diagnostic agent. After allowing the labeled antibody to
bind to CEA-expressing cells, the distribution of bound antibody may be
imaged or otherwise determined. For treatment of CEA-expressing tumors,
the Class I anti-CEA antibody may be conjugated to at least one
therapeutic agent and the immunoconjugate administered to a patient.

[0019] In alternative embodiments, the Class I anti-CEA antibody may be
part of a bispecific or multispecific antibody. Such antibodies may
contain at least one binding site for a tumor-associated antigen, such as
CEA, and at least one other binding site for a hapten attached to a
targetable construct. Such bispecific or multispecific antibodies may be
used in pretargeting methods for diagnosis or treatment of cancer, as
discussed in more detail below. Where pretargeting is used, the
bispecific or multispecific antibody may be administered to a subject and
allowed to localize to a CEACAM5- or CEACAM6-expressing tumor. A clearing
agent may optionally be added to enhance clearance of unbound antibody
from the circulation. After allowing a sufficient time for unbound
antibody to clear from the circulation, a targetable construct conjugated
to a therapeutic and/or diagnostic agent may be administered to the
subject to bind to the antibody localized at the tumor site. Delivery of
diagnostic agents using a Class I anti-CEA antibody may be performed as
part of an endoscopic, intravascular or intraoperative procedure.

[0020] In various embodiments, the therapeutic agent is selected from the
group consisting of a naked antibody, a cytotoxic agent, a drug, a
radionuclide, boron atoms, an immunomodulator, a photoactive therapeutic
agent, an immunoconjugate, a hormone, an enzyme, an antisense
oligonucleotide or a combination thereof, optionally formulated in a
pharmaceutically acceptable vehicle. Preferably, the therapeutic agent is
a cytotoxic agent selected from a drug or a toxin. It is contemplated
that the drug may possess a pharmaceutical property selected from the
group consisting of antimitotic, alkylating, antimetabolite,
antiangiogenic, apoptotic, alkaloid, COX-2, and antibiotic agents and
combinations thereof. Preferably, the drug is selected from the group
consisting of nitrogen mustards, ethylenimine derivatives, alkyl
sulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines,
taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs,
antimetabolites, antibiotics, epipodophyllotoxins, platinum coordination
complexes, vinca alkaloids, substituted ureas, methyl hydrazine
derivatives, adrenocortical suppressants, antagonists, endostatin,
taxols, camptothecins, oxaliplatin, doxorubicins and their analogs.
Exemplary oligonucleotides may include siRNA or RNAi molecules. Many
examples of therapeutic oligonucleotides are known in the art and any
such known example may be attached to a subject Class I anti-CEA antibody
or fragment thereof.

[0022] In another embodiment, an immunomodulator is administered prior to
the administration of a therapeutically effective amount of a Class I
anti-CEA monoclonal antibody or fragment thereof alone or a Class I
anti-CEA monoclonal antibody and at least one therapeutic agent.
Immunomodulators may be selected from the group consisting of a cytokine,
a stem cell growth factor, a lymphotoxin, an hematopoietic factor, a
colony stimulating factor (CSF), an interferon (IFN), a stem cell growth
factor, erythropoietin, thrombopoietin and a combination thereof.
Preferably, the lymphotoxin is tumor necrosis factor (TNF), the
hematopoietic factor is an interleukin (IL), the colony stimulating
factor is granulocyte-colony stimulating factor (G-CSF) or granulocyte
macrophage-colony stimulating factor (GM-CSF)), the interferon is
interferon-α, -β or -γ, and the stem cell growth factor
is designated "S1 factor."

[0027] In various embodiments, the bispecific or multispecific antibodies
or other antibody constructs may be produced as fusion proteins or by use
of the Dock-and-Lock (DNL) technology, as described in more detail below.
Compositions and methods for production and use of DNL constructs have
been reported (see, e.g., U.S. Pat. Nos. 7,521,056; 7,527,787; 7,534,866;
7,550,143 and 7,666,400 and U.S. patent application Ser. Nos. 12/418,877;
12/544,476; 12/731,781; 12/752,649; and 12/754,740, the Examples section
of each of which is incorporated herein by reference). DNL complexes are
formed by attachment of a selected effector to an anchoring domain (AD)
or dimerization and docking domain (DDD) peptide sequence. The DNL
complex forms when the DDD sequence spontaneously dimerizes and binds to
the AD sequence. Virtually any effector moiety may be attached to a DDD
or AD sequence, including antibodies or antibody fragments, peptides,
proteins, enzymes, toxins, therapeutic agents, diagnostic agents,
immunomodulators, polymers such as polyethylene glycol (PEG), cytokines,
chemokines, growth factors, hormones and any other type of molecule or
complex. In preferred embodiments, the DNL complex may comprise an
antibody or fragment thereof comprising light chain variable region CDR
sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT
(SEQ ID NO:3); and heavy chain variable region CDR sequences DYYMS (SEQ
ID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID
NO:6). In other preferred embodiments, the DNL complex may further
comprise a cytokine or a polyethylene glycol moiety. These embodiments
are not limiting and the skilled artisan will realize that the claimed
DNL complexes may comprise a Class I anti-CEA antibody moiety attached to
virtually any other effector moiety.

[0032]FIG. 5 shows a schematic structural drawing of the domains of
CEACAM5 (left) and CEACAM 6 (right). The epitopes recognized by MN-15,
MN-3 and MN-14 antibodies on CEACAM5 and CEACAM6 are as noted.

[0033]FIG. 6 shows the results of an in vitro endothelial cell adhesion
assay. Percentage adhesion of various tumor cells with varying amounts of
expressed CEACAM5 and CEACAM6 to HUVEC cells in the absence or presence
of MN-15 Fab', MN-3 Fab', or Ag8 Fab' control. Cells were labeled with 1
μCi/mL of 3H-thymidine and added to HUVEC cultures and incubated
for 30 minutes at 37° C. Samples were washed thrice with PBS to
remove unattached cells. Attached tumor cells were solubilized with 0.1 N
NaOH and radioactivity was measured in a β-scintillation counter.
The cpm attached/total cpm added (attaching potential) was determined.
Results of a typical study are presented. Cell lines used include BT-20
(CEACAM5+/CEACAM6+), MCF-7 (CEACAM5-/CEACAM6+), HT-29
(CEACAM5-/CEACAM6+), Moser (CEACAM5+/CEACAM6-), MCA38cea
(CEACAM5+/CEACAM6-), and MCA38 (CEACAM5-/CEACAM6-). Both MN-15 and MN-3
induced a 49% to 58% inhibition in adhesion in four cell lines (P<0.01
for MN-3 Fab' on MCF-7, HT-29, and BT-20; P<0.02 for MN-15 on the same
three lines; and P<0.05 for both Fabs on Moser adhesion to endothelial
cells).

[0034] FIG. 7 illustrates an exemplary in vivo micrometastasis study. Top,
immunohistochemistry of GW-39 tumor sections stained with a nonspecific
antibody (A), MN-14 anti-CEACAM5 (B), and MN-15 anti-CEACAM6 (C) and
photographed at 100×. Bottom, survival of nude mice implanted with
30 μL of a 10% suspension of GW-39 human colonic cancer cells. Mice
were implanted with cells that were preincubated for 30 minutes with
MN-3-, MN-15-, or hMN-14-Fab' (10 μg/mL). Mice also received a single
100 μg dose of the same Fab' 1 day after cell implantation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention provides methods of treatment in which a
chimeric, humanized or human Class I anti-CEA antibody or fragment
thereof is administered to a subject with cancer. The methods are of use
to treat cancers that express CEACAM5 (CEA) and/or CEACAM6, since Class I
anti-CEA antibodies bind to both antigens. The antibodies may be
administered as naked (unconjugated) antibodies or as immunoconjugates
attached to one or more therapeutic agents. The naked antibodies may be
administered prior to, simultaneously with or after one or more other
therapeutic anti-cancer agents.

[0036] The method is useful for treating a wide variety of cancers,
including but not limited to medullary thyroid carcinoma, colorectal
cancers, hepatocellular carcinoma, pancreatic, breast, lung,
head-and-neck, bladder, uterine and ovarian cancers, and even cancers
that do not express CEACAM5 or CEACAM6 at very high levels. For example,
treatment is contemplated in cancers that express CEA at levels of at
least 100 ng/g of tissue.

[0038] The mechanism of tumor cell killing by the naked Class I anti-CEA
antibody is not known with certainty and likely involves several
mechanisms. It is hypothesized that the naked antibody alone or in
combination with a therapeutic agent may affect tumor growth by blocking
biological activities of their respective antigen or by stimulating
natural immunological functions, such as antibody-dependent cell-mediated
cytotoxicity (ADCC) or complement-mediated lysis. Additionally, the naked
antibody alone or in combination with the therapeutic agent may treat and
control the cancer by inhibiting cell growth and cell cycle progression,
inducing apoptosis, inhibiting angiogenesis, inhibiting metastatic
activity, and/or affecting tumor cell adhesion. In fact, the anti-CEA
antibody or fragment thereof may be more effective in treating metastases
than primary cancers, since the metastases may be more susceptible to
antagonists of tumor cell adhesion. The present treatment method provides
a treatment plan that may be optimized to provide the maximum anti-tumor
activity for individual patients by allowing the titration of the
antibody and one or more different therapeutic agents to provide an
effective treatment regimen.

[0039] In certain alternative embodiments, a preferred naked murine,
chimeric, humanized or human Class I anti-CEA antibody may be a
monovalent construct, comprising only one binding site for CEACAM5 or
CEACAM6. For example, a Fab, Fab' or scFv antibody fragment comprising
light chain variable region CDR sequences SASSRVSYIH (SEQ ID NO:1);
GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy chain
variable region CDR sequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG
(SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6) may be utilized. Such
monovalent constructs may be conjugated to a polymer, such as PEG, to
extend its serum half-life, using techniques described in detail below.
Another alternative monovalent antibody fragment may contain a human IgG4
constant region and hinge. In certain embodiments, the native IgG4
sequence may be modified by replacing cysteine residues with serine
residues, as discussed in McDonagh et al., (2006, Protein Eng Des Sel
19:299-307). In still other alternative embodiments, a monovalent
antibody fragment may be constructed by the DNL technique, also described
in detail below.

DEFINITIONS

[0040] As used herein, the terms "a", "an" and "the" may refer to either
the singular or plural, unless the context otherwise makes clear that
only the singular is meant.

[0041] An antibody, as described herein, refers to a full-length (i.e.,
naturally occurring or formed by normal immunoglobulin gene fragment
recombinatorial processes) immunoglobulin molecule (e.g., an IgG
antibody) or an immunologically active (i.e., specifically binding)
portion of an immunoglobulin molecule, like an antibody fragment.

[0042] An antibody fragment is a portion of an antibody such as
F(ab')2, Fab', Fab, Fv, scFv (single chain Fv) and the like.
Regardless of structure, an antibody fragment binds with the same antigen
that is recognized by the intact antibody. The term "antibody fragment"
includes isolated fragments consisting of the variable regions, such as
the "Fv" fragments consisting of the variable regions of the heavy and
light chains, recombinant single chain polypeptide molecules in which
light and heavy variable regions are connected by a peptide linker ("scFv
proteins"), and minimal recognition units consisting of the amino acid
residues that mimic the hypervariable region. The Fv fragments may be
constructed in different ways as to yield multivalent and/or
multispecific binding forms. In the former case of multivalent, they
react with more than one binding site against the CEA epitope, whereas
with multispecific forms, more than one epitope (either of CEA or even
against CEA and a different antigen) is bound.

[0043] A naked antibody is generally an entire antibody which is not
conjugated to a therapeutic agent. This is so because the Fc portion of
the antibody molecule provides effector or immunological functions, such
as complement fixation and ADCC (antibody dependent cell cytotoxicity).
However, the Fc portion may not be required for therapeutic function of
the antibody, but rather other mechanisms, such as apoptosis,
anti-angiogenesis, anti-metastatic activity, anti-adhesion activity, such
as inhibition of heterotypic or homotypic adhesion, and interference in
signaling pathways, may come into play and interfere with the disease
progression. Naked antibodies include both polyclonal and monoclonal
antibodies, and fragments thereof, that include murine antibodies, as
well as certain recombinant antibodies, such as chimeric, humanized or
human antibodies and fragments thereof. As defined herein, "naked" is
synonymous with "unconjugated," and means not linked or conjugated to any
therapeutic agent.

[0044] A chimeric antibody is a recombinant protein that contains the
variable domains of the heavy and light antibody chains, including the
complementarity determining regions (CDRs) of an antibody derived from
one species, preferably a rodent antibody, while the constant domains of
the antibody molecule are derived from those of a human antibody.

[0045] A humanized antibody is a recombinant protein in which the CDRs
from an antibody from one species; e.g., a rodent antibody, are
transferred from the heavy and light variable chains of the rodent
antibody into human heavy and light variable domains. The constant
domains of the antibody molecule are derived from those of a human
antibody.

[0046] A human antibody is an antibody obtained from transgenic mice that
have been "engineered" to produce specific human antibodies in response
to antigenic challenge. In this technique, elements of the human heavy
and light chain locus are introduced into strains of mice derived from
embryonic stem cell lines that contain targeted disruptions of the
endogenous heavy chain and light chain loci. The transgenic mice can
synthesize human antibodies specific for human antigens, and the mice can
be used to produce human antibody-secreting hybridomas. Methods for
obtaining human antibodies from transgenic mice are described by Green et
al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994),
and Taylor et al., Int. Immun. 6:579 (1994). A fully human antibody also
can be constructed by genetic or chromosomal transfection methods, as
well as phage display technology, all of which are known in the art. See
for example, McCafferty et al., Nature 348:552-553 (1990) for the
production of human antibodies and fragments thereof in vitro, from
immunoglobulin variable domain gene repertoires from unimmunized donors.
In this technique, antibody variable domain genes are cloned in-frame
into either a major or minor coat protein gene of a filamentous
bacteriophage, and displayed as functional antibody fragments on the
surface of the phage particle. Because the filamentous particle contains
a single-stranded DNA copy of the phage genome, selections based on the
functional properties of the antibody also result in selection of the
gene encoding the antibody exhibiting those properties. In this way, the
phage mimics some of the properties of the B cell. Phage display can be
performed in a variety of formats, for their review, see e.g. Johnson and
Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993). Human
antibodies may also be generated by in vitro activated B cells. See U.S.
Pat. Nos. 5,567,610 and 5,229,275, which are incorporated in their
entirety by reference.

[0047] A therapeutic agent is a molecule or atom which is administered
separately, concurrently or sequentially with an antibody or antibody
fragment, and is useful in the treatment of a disease. Examples of
therapeutic agents include antibodies, antibody fragments,
immunoconjugates, drugs, enzymes, cytotoxic agents, toxins, nucleases,
hormones, immunomodulators, chelators, boron compounds, photoactive
agents or dyes, radioisotopes or radionuclides, antisense
oligonucleotides or combinations thereof.

[0048] As used herein, the term antibody fusion protein is a recombinantly
produced antigen-binding molecule in which one or more of the same or
different natural antibody, single-chain antibody or antibody fragment
segments with the same or different specificities are linked. A Class I
anti-CEA fusion protein comprises at least one CEA binding site.
Preferably, the Class I anti-CEA fusion protein comprises the light chain
variable region CDR sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID
NO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy chain variable region CDR
sequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and
DMGIRWNFDV (SEQ ID NO:6). Valency of the fusion protein indicates the
total number of binding arms or sites the fusion protein has to
antigen(s) or epitope(s); i.e., monovalent, bivalent, trivalent or
mutlivalent. The multivalency of the antibody fusion protein means that
it can take advantage of multiple interactions in binding to an antigen,
thus increasing the avidity of binding to the antigen, or to different
antigens. Specificity indicates how many different types of antigen or
epitope an antibody fusion protein is able to bind; i.e., monospecific,
bispecific, trispecific, multispecific. Using these definitions, a
natural antibody, e.g., an IgG, is bivalent because it has two binding
arms but is monospecific because it binds to one type of antigen or
epitope. In certain embodiments, a fusion protein may comprise one or
more antibodies or fragments thereof linked to a different effector
protein or peptide, such as a cytokine, hormone, growth factor, binding
protein, binding peptide or other effector. An exemplary fusion protein
may comprise an antibody or fragment thereof attached to an AD or DDD
peptide, as discussed in detail below.

[0049] An immunomodulator is a therapeutic agent that when present,
alters, suppresses or stimulates the body's immune system. Typically, an
immunomodulator of use acts to stimulate immune cells to proliferate or
become activated in an immune response cascade, such as macrophages,
B-cells, and/or T-cells. An example of an immunomodulator as described
herein is a cytokine, which is a soluble small protein of approximately
5-20 kDa that is released by one cell population (e.g., primed
T-lymphocytes) on contact with specific antigens, and which acts as an
intercellular mediator between cells. As the skilled artisan will
understand, examples of cytokines include lymphokines, monokines,
interleukins, and several related signalling molecules, such as tumor
necrosis factor (TNF) and interferons. Chemokines are a subset of
cytokines. Certain interleukins and interferons are examples of cytokines
that stimulate T cell or other immune cell proliferation.

[0051] Monoclonal antibodies (MAbs) to specific antigens may be obtained
by methods known to those skilled in the art. See, for example, Kohler
and Milstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENT
PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons
1991) [hereinafter "Coligan"]. Briefly, monoclonal antibodies can be
obtained by injecting mice with a composition comprising an antigen,
verifying the presence of antibody production by removing a serum sample,
removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes
with myeloma cells to produce hybridomas, cloning the hybridomas,
selecting positive clones which produce antibodies to the antigen,
culturing the clones that produce antibodies to the antigen, and
isolating the antibodies from the hybridoma cultures.

[0053] MAbs to peptide backbones are generated by well-known methods for
Ab production. For example, injection of an immunogen, such as
(peptide)N-KLH, wherein KLH is keyhole limpet hemocyanin, and
N=1-30, in complete Freund's adjuvant, followed by two subsequent
injections of the same immunogen suspended in incomplete Freund's
adjuvant into immunocompetent animals. The animals are given a final i.v.
boost of antigen, followed by spleen cell harvesting three days later.
Harvested spleen cells are then fused with Sp2/0-Ag14 myeloma cells and
culture supernatants of the resulting clones analyzed for anti-peptide
reactivity using a direct binding ELISA. Fine specificity of generated
MAbs can be analyzed for by using peptide fragments of the original
immunogen. These fragments can be prepared readily using an automated
peptide synthesizer. For MAb production, enzyme-deficient hybridomas are
isolated to enable selection of fused cell lines. This technique also can
be used to raise antibodies to one or more chelating or other hapten
moieties, such as In(III)-DTPA chelates. Monoclonal mouse antibodies to
an In(III)-di-DTPA are known (U.S. Pat. No. 5,256,395 to Barbet).

[0054] Another method for producing antibodies is by production in the
milk of transgenic livestock. See, e.g., Colman, A., Biochem. Soc. Symp.,
63: 141-147, 1998; U.S. Pat. No. 5,827,690, both of which are
incorporated in their entirety by reference. Two DNA constructs are
prepared which contain, respectively, DNA segments encoding paired
immunoglobulin heavy and light chains. The DNA segments are cloned into
expression vectors that contain a promoter sequence that is
preferentially expressed in mammary epithelial cells. Examples include,
but are not limited to, promoters from rabbit, cow and sheep casein
genes, the cow α-lactoglobulin gene, the sheep β-lactoglobulin
gene and the mouse whey acid protein gene. Preferably, the inserted
fragment is flanked on its 3' side by cognate genomic sequences from a
mammary-specific gene. This provides a polyadenylation site and
transcript-stabilizing sequences. The expression cassettes are coinjected
into the pronuclei of fertilized, mammalian eggs, which are then
implanted into the uterus of a recipient female and allowed to gestate.
After birth, the progeny are screened for the presence of both transgenes
by Southern analysis. In order for the antibody to be present, both heavy
and light chain genes must be expressed concurrently in the same cell.
Milk from transgenic females is analyzed for the presence and
functionality of the antibody or antibody fragment using standard
immunological methods known in the art. The antibody can be purified from
the milk using standard methods known in the art.

[0055] After the initial raising of antibodies to the immunogen, the
variable genes of the monoclonal antibodies can be cloned from the
hybridoma cells, sequenced and subsequently prepared by recombinant
techniques. General techniques for cloning murine immunoglobulin variable
domains are described, for example, by the publication of Orlandi et al.,
Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Humanization and
chimerization of murine antibodies and antibody fragments are well known
to those skilled in the art. A chimeric antibody is a recombinant protein
that contains the variable domains including the CDRs derived from one
species of animal, such as a rodent antibody, while the remainder of the
antibody molecule; i.e., the constant domains, is derived from a human
antibody. The use of antibody components derived from humanized and
chimeric monoclonal antibodies alleviates potential problems associated
with the immunogenicity of murine constant regions. Techniques for
constructing chimeric antibodies are well known to those of skill in the
art. As an example, Leung et al., Hybridoma 13:469 (1994), describe how
they produced an LL2 chimera by combining DNA sequences encoding the
VK and VH domains of the murine LL2 monoclonal antibody, an
anti-CD22 antibody, with respective human K and IgG1 constant region
domains.

[0056] A chimeric monoclonal antibody (MAb) can be humanized by replacing
the sequences of the murine FR in the variable domains of the chimeric
MAb with one or more different human FR. As simply transferring mouse
CDRs into human FRs often results in a reduction or even loss of antibody
affinity, additional modification might be required in order to restore
the original affinity of the murine antibody. This can be accomplished by
the replacement of one or more human residues in the FR regions with
their murine counterparts to obtain an antibody that possesses good
binding affinity to its epitope. See, for example, Tempest et al.,
Biotechnology 9:266 (1991) and Verhoeyen et al., Science 239: 1534
(1988).

[0057] Additionally, knowing that chimeric anti-CEA exhibits a binding
affinity comparable to that of its murine counterpart, defective designs,
if any, in the original version of the humanized anti-CEA MAb can be
identified by mixing and matching the light and heavy chains of the
chimeric anti-CEA to those of the humanized version. Preferably, the
humanized anti-CEA antibody comprises the light chain variable region CDR
sequences CDR1 having an amino acid sequence SASSRVSYIH (SEQ ID NO:1);
CDR2 having an amino acid sequence GTSTLAS (SEQ ID NO:2); and CDR3 having
an amino acid sequence QQWSYNPPT (SEQ ID NO:3); and the heavy chain
variable region CDR sequences CDR1 having an amino acid sequence DYYMS
(SEQ ID NO:4); CDR2 having an amino acid sequence FIANKANGHTTDYSPSVKG
(SEQ ID NO:5); and CDR3 having an amino acid sequence DMGIRWNFDV (SEQ ID
NO:6). More preferably, the humanized anti-CEA antibody comprises the
light chain FR sequences of the human REI antibody and the heavy chain FR
sequences of the human KOL antibody. Most preferably, the humanized
anti-CEA antibody comprises one or more murine FR amino acid residues
selected from the heavy chain amino acid residues 28, 29, 30, 48 and 49
of SEQ ID NO:10 and light chain amino acid residues 21, 47 and 60 of SEQ
ID NO:9.

[0058] Alternatively, a combination of framework sequences from 2 or more
different human antibodies can be used for VH and VK FR
sequences. The production of humanized MAbs is described, for example, by
Jones et al., Nature 321:522 (1986), Riechmann et al., Nature 332:323
(1988), Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc.
Nat'l Acad. Sci. USA 89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437
(1992), and Singer et al., J. Immun. 150:2844 (1993). Further, the
affinity of humanized, chimeric and human MAbs to a specific epitope can
be increased by mutagenesis of the CDRs, so that a lower dose of antibody
may be as effective as a higher dose of a lower affinity MAb prior to
mutagenesis. See for example, WO0029584A1.

[0059] In another embodiment, a Class I anti-CEA monoclonal antibody is a
human antibody. The human anti-CEA MAb, or another human antibody, can be
obtained from a transgenic non-human animal. See, e.g., Mendez et al.,
Nature Genetics, 15: 146-156 (1997) and U.S. Pat. No. 5,633,425. For
example, a human antibody can be recovered from a transgenic mouse
possessing human immunoglobulin loci. The mouse humoral immune system is
humanized by inactivating the endogenous immunoglobulin genes and
introducing human immunoglobulin loci. The human immunoglobulin loci are
exceedingly complex and comprise a large number of discrete segments
which together occupy almost 0.2% of the human genome. To ensure that
transgenic mice are capable of producing adequate repertoires of
antibodies, large portions of human heavy- and light-chain loci must be
introduced into the mouse genome. This is accomplished in a stepwise
process beginning with the formation of yeast artificial chromosomes
(YACs) containing either human heavy- or light-chain immunoglobulin loci
in germline configuration. Since each insert is approximately 1 Mb in
size, YAC construction requires homologous recombination of overlapping
fragments of the immunoglobulin loci. The two YACs, one containing the
heavy-chain loci and one containing the light-chain loci, are introduced
separately into mice via fusion of YAC containing yeast spheroblasts with
mouse embryonic stem cells. Embryonic stem cell clones are then
microinjected into mouse blastocysts. Resulting chimeric males are
screened for their ability to transmit the YAC through their germline and
are bred with mice deficient in murine antibody production. Breeding the
two transgenic strains, one containing the human heavy-chain loci and the
other containing the human light-chain loci, creates progeny which
produce human antibodies in response to immunization.

[0061] As an alternative, human antibody fragments may be isolated from a
combinatorial immunoglobulin library. See, e.g., Barbas et al., METHODS:
A Companion to Methods in Enzymology 2: 119 (1991), and Winter et al.,
Ann. Rev. Immunol. 12: 433 (1994). Many of the difficulties associated
with generating monoclonal antibodies by B-cell immortalization can be
overcome by engineering and expressing antibody fragments in E. coli,
using phage display. To ensure the recovery of high affinity, monoclonal
antibodies a combinatorial immunoglobulin library must contain a large
repertoire size. A typical strategy utilizes mRNA obtained from
lymphocytes or spleen cells of immunized mice to synthesize cDNA using
reverse transcriptase. The heavy- and light-chain genes are amplified
separately by PCR and ligated into phage cloning vectors. Two different
libraries are produced, one containing the heavy-chain genes and one
containing the light-chain genes. Phage DNA is isolated from each
library, and the heavy-and light-chain sequences are ligated together and
packaged to form a combinatorial library. Each phage contains a random
pair of heavy- and light-chain cDNAs and upon infection of E. coli
directs the expression of the antibody chains in infected cells. To
identify an antibody that recognizes the antigen of interest, the phage
library is plated, and the antibody molecules present in the plaques are
transferred to filters. The filters are incubated with radioactively
labeled antigen and then washed to remove excess unbound ligand. A
radioactive spot on the autoradiogramidentifies a plaque that contains an
antibody that binds the antigen. Cloning and expression vectors that are
useful for producing a human immunoglobulin phage library can be
obtained, for example, from STRATAGENE Cloning Systems (La Jolla,
Calif.).

[0062] In one embodiment, the antibodies are produced as described in
Hansen et al., Cancer, 71:3478 (1993); Hansen et al., U.S. Pat. No.
5,874,540; Primus et al., U.S. Pat. No. 4,818,709, and Shively et al.,
U.S. Pat. No. 5,081,235, the examples section of each of which is
incorporated herein by reference.

[0063] Production of Antibody Fragments

[0064] Certain embodiments concern the use of fragments of a Class I
anti-CEA antibody, preferably comprising light chain variable region CDR
sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT
(SEQ ID NO:3); and heavy chain variable region CDR sequences DYYMS (SEQ
ID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID
NO:6). Antibody fragments which recognize the same epitope as a parent
antibody can be generated by known techniques. For example, antibody
fragments can be prepared by proteolytic hydrolysis of an antibody or by
expression in E. coli of the DNA coding for the fragment. The antibody
fragments are antigen binding portions of an antibody, such as
F(ab')2, Fab', Fab, Fv, scFv and the like, and can be obtained by
pepsin or papain digestion of whole antibodies by conventional methods or
by genetic engineering techniques.

[0065] For example, an antibody fragment can be produced by enzymatic
cleavage of antibodies with pepsin to provide a 100 Kd fragment denoted
F(ab')2. This fragment can be further cleaved using a thiol reducing
agent, and optionally a blocking group for the sulfhydryl groups
resulting from cleavage of disulfide linkages, to produce 50 Kd Fab'
monovalent fragments. Alternatively, an enzymatic cleavage using papain
produces two monovalent Fab fragments and an Fc fragment directly. These
methods are described, for example, by Goldenberg, U.S. Pat. Nos.
4,036,945 and 4,331,647 and references contained therein. Also, see
Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem.
J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. I, page
422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and
2.10.-2.10.4.

[0066] Other methods of cleaving antibodies, such as separation of heavy
chains to form monovalent light-heavy chain fragments, further cleavage
of fragments, or other enzymatic, chemical or genetic techniques may also
be used, so long as the fragments bind to the antigen that is recognized
by the intact antibody.

[0067] For example, Fv fragments comprise an association of VH and
VL chains. This association can be noncovalent, as described in
Inbar et al., Proc. Nat'l. Acad. Sci. U.S.A. 69:2659 (1972).
Alternatively, the variable chains can be linked by an intermolecular
disulfide bond or cross-linked by chemicals such as glutaraldehyde. See,
for example, Sandhu, Crit. Rev. Biotech. 12:437 (1992). Preferably, the
Fv fragments comprise VH and VL chains which are connected by a
peptide linker. These single-chain antigen binding proteins (scFv) are
prepared by constructing a structural gene comprising DNA sequences
encoding the VH and VL domains which are connected by an
oligonucleotide. The structural gene is inserted into an expression
vector that is subsequently introduced into a host cell, such as E. coli.
The recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing scFvs
are described, for example, by Whitlow et al., Methods: A Companion to
Methods in Enzymology, 2:97 (1991). Also see Bird et al., Science 242:423
(1988), Ladner et al., U.S. Pat. No. 4,946,778; Pack et al., Bio
Technology 11: 1271 (1993) and Sandhu, supra.

[0068] Another form of an antibody fragment is a peptide coding for a
single complementarity-determining region (CDR). A CDR is a segment of
the variable region of an antibody that is complementary in structure to
the epitope to which the antibody binds and is more variable than the
rest of the variable region. Accordingly, a CDR is sometimes referred to
as hypervariable region. A variable region comprises three CDRs. CDR
peptides can be obtained by constructing genes encoding the CDR of an
antibody of interest. Such genes are prepared, for example, by using the
polymerase chain reaction to synthesize the variable region from RNA of
antibody producing cells. See, for example, Larrick et at., Methods: A
Companion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,
"Genetic Manipulation of Monoclonal Antibodies," in MONOCLONAL
ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et
al. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et
al., "Genetic Manipulation and Expression of Antibodies," in MONOCLONAL
ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages
137-185 (Wiley-Liss, Inc. 1995).

[0069] Other antibody fragments, for example single domain antibody
fragments, are known in the art and may be used in the claimed
constructs. Single domain antibodies (VHH) may be obtained, for example,
from camels, alpacas or llamas by standard immunization techniques. (See,
e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol
Methods 281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25,
2007). The VHH may have potent antigen-binding capacity and can interact
with novel epitopes that are inacessible to conventional VH-VL pairs.
(Muyldermans et al., 2001). Alpaca serum IgG contains about 50% camelid
heavy chain-only IgG antibodies (HCAbs) (Maass et al., 2007). Alpacas may
be immunized with known antigens, such as TNF-α, and VHHs can be
isolated that bind to and neutralize the target antigen (Maass et al.,
2007). PCR primers that amplify virtually all alpaca VHH coding sequences
have been identified and may be used to construct alpaca VHH phage
display libraries, which can be used for antibody fragment isolation by
standard biopanning techniques well known in the art (Maass et al.,
2007).

[0072] A composition as claimed herein may comprise at least one Class I
anti-CEA monoclonal antibody (MAb) or fragment thereof. In certain
embodiments, the composition or method of use may also include at least
one therapeutic agent, which may be conjugated to the Class I anti-CEA
antibody or not conjugated. In compositions comprising more than one
antibody or antibody fragments, such as a second Class I anti-CEA
antibody, the second antibody is non-blocking (i.e., does not block
binding of the first Class I anti-CEA antibody or antibody fragment to
its target antigen).

[0076] Also described herein is a composition comprising a naked murine,
humanized, chimeric or human Class I anti-CEA antibody or fragment
thereof. The composition may optionally comprise a therapeutic agent,
such as a second naked or conjugated anti-CEA antibody or antibody
fragment thereof. Where a second anti-CEA antibody is used, it is
non-blocking, i.e., does not block binding of the first Class I anti-CEA
antibody or fragment thereof. In other words, both anti-CEA antibodies or
fragments thereof are non-blocking to each other, allowing both
antibodies or fragments thereof to bind to CEA (CD66e).

[0077] In still other embodiments, the claimed compositions and methods
may comprise an antibody or fragment that binds to the same epitope of
CEACAM5 or CEACAM6 as a Class I anti-CEA antibody comprising light chain
variable region CDR sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID
NO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy chain variable region CDR
sequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and
DMGIRWNFDV (SEQ ID NO:6). Evidence of epitope binding may be determined,
for example, by competitive binding assays that are well known in the
art. Additionally, other anti-CEA antibodies, such as Class II or Class
III anti-CEA antibodies, can be used in combination with the Class I
anti-CEA antibody, in either a naked or conjugated form. For example, one
or more chimeric or humanized Class II or Class III anti-CEA antibodies
or fragments thereof may be combined with a Class I anti-CEA antibody or
fragment thereof.

[0079] The second antibody or antibody fragment may be either unconjugated
(naked) or conjugated to at least one therapeutic agent (immunocougate).
Immunoconjugates can be prepared by indirectly conjugating a therapeutic
agent to an antibody component. General techniques are described in Shih
et al., Int. J. Cancer, 41:832 (1988); Shih et al., Int. J. Cancer,
46:1101 (1990); and Shih et al., U.S. Pat. No. 5,057,313. The general
method involves reacting an antibody component having an oxidized
carbohydrate portion with a carrier polymer that has at least one free
amine function and that is loaded with a plurality of drug, toxin,
chelator, boron addends, or other therapeutic agents. This reaction
results in an initial Schiff base (imine) linkage, which can be
stabilized by reduction to a secondary amine to form the final conjugate.
However, as discussed below many methods of preparing immunoconjugates
are known in the art and any such known method may be used.

[0082] Also described herein are methods for treating carcinomas.
Exemplary carcinomas include medullary thyroid carcinoma, colorectal
cancer, pancreatic cancer, breast cancer, hepatocellular carcinoma,
ovarian cancer, gastric cancer, prostate cancer, uterine cancer,
hematopoietic cancer, leukemia and various lung, head-and-neck,
endometrial, bladder, and liver cancers that express CEACAM5 and/or
CEACAM6. The CEA levels in these types of cancers are lower than present
in medullary thyroid carcinomas but all that is necessary is that the
CEACAM5 and/or CEACAM6 levels be sufficiently high so that the Class I
anti-CEA therapy provides an effective treatment. Normal colon mucosa has
about 100-500 ng/gram but carcinomas expressing CEA at levels of about 5
mcg/gram of tissue are suitable for treatment with the methods described
herein.

[0083] For example, contemplated herein is a method for treating cancer
comprising administering to a subject, either concurrently or
sequentially, a therapeutically effective amount of a Class I anti-CEA
monoclonal antibody or fragment thereof and at least one therapeutic
agent, optionally formulated in a pharmaceutically acceptable vehicle.
Preferably, the Class I anti-CEA monoclonal antibody or fragment thereof
is chimeric, murine, humanized or human, wherein the Class I anti-CEA MAb
retains substantially the Class I anti-CEA binding specificity of a
parental murine MAb. More preferably, the Class I anti-CEA antibody
comprises light chain variable region CDR sequences SASSRVSYIH (SEQ ID
NO:1); GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy
chain variable region CDR sequences DYYMS (SEQ ID NO:4);
FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6).
Preferably the therapeutic agent is a cytotoxic agent, more preferably an
alkylating agent, and most preferably, dacarbazine (DTIC). But in another
embodiment, the therapeutic agent may not be DTIC. Other classes of
anti-cancer cytostatic and cytotoxic agents, such as 5-fluorouracil,
CPT-11 and oxaliplatin can also be used in combinations with these
antibodies, especially in the therapy of colorectal cancers. In other
cancer types, cancer drugs that are known to be effective are also good
candidates for combining with the antibody therapies proposed herein.

[0085] A naked Class I anti-CEA antibody as described herein can
significantly increase the chemosensitivity of cancer cells to one or
more therapeutic agents. For example, treatment of colon cancer cells
with a naked Class I, anti-CEA antibody comprising light chain variable
region CDR sequences SASSRVSYIH (SEQ ID NO:1); GTSTLAS (SEQ ID NO:2); and
QQWSYNPPT (SEQ ID NO:3); and heavy chain variable region CDR sequences
DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG (SEQ ID NO:5); and DMGIRWNFDV
(SEQ ID NO:6), as described herein, either before or concurrently with a
therapeutic agent, such as DTIC, CPT-11,5'-fluorouracil (5-FU) or
oxaliplatin, improves a cell's response to a therapeutic agent, such as a
cytotoxic drug. Further, these therapeutic methods of treatment with a
naked Class I, anti-CEA antibody alone or in combination with a
therapeutic agent can be further enhanced by administering an
immunomodulator as described herein, prior to the administration of the
naked antibody or the administration of the naked antibody and at least
one of the therapeutic agents.

[0087] Preferably, a therapeutic agent of use is a drug used in standard
cancer chemotherapy, such as taxane or platinum drugs in ovarian cancer,
fluorouracil, CPT-11, and oxaloplatin drugs in colorectal cancer,
gemcitabine in pancreatic and other cancers, or taxane derivatives in
breast cancers. COX-2 inhibitors represent still another class of agents
that show activity in combination with typical cytotoxic agents in cancer
chemotherapy, and can be used in the same way, but combined in addition
with anti-CEA antibodies alone or in combination with other anti-TAA
antibodies. Optionally, these drugs can be used in combination with
radiolabeled antibodies, either anti-CEA antibody conjugates or
radioimmunoconjugates with other anti-TAA antibodies.

[0088] In a preferred embodiment, a naked Class I anti-CEA antibody or
fragment thereof is administered sequentially (either prior to or after)
or concurrently with dacarbazine (DTIC), doxorubin, cyclophosphamide or
vincristine, or any combination of these. For example, DTIC and
cyclophosphamide may be administered sequentially or concurrently with a
naked Class I anti-CEA antibody or fragment thereof. Similarly,
5-fluorouracil in combination with folinic acid, alone or in combination
with irinotecan (CPT-11) or oxaliplatin, is a regimen used to treat
colorectal cancer. Other suitable combination chemotherapeutic regimens
are well known, such as with oxaliplatin alone, or in combination with
these other drugs, to those of skill in the art. Accordingly, combination
therapy with any of these chemotherapeutic agents and a naked Class I
anti-CEA antibody or fragment thereof can be used to treat cancer. In
medullary thyroid carcinoma, other chemotherapeutic agents may be
preferred, such as one of the alkylating agents (e.g., DTIC), as well as
gemcitabine and other more recent classes of cytotoxic drugs. The
chemotherapeutic drugs and a naked Class I anti-CEA antibody or fragment
thereof, can be administered in any order, or together. In a preferred
multimodal therapy, both chemotherapeutic drugs and naked Class I
anti-CEA antibodies or fragments thereof are administered before, after,
or co-administered with a conjugated or unconjugated anti-CEA antibody,
fusion protein, or fragment thereof. Preferably, the Class I anti-CEA
antibody or fragment thereof is a humanized antibody or fragment thereof.

[0089] A preferred treatment schedule of multimodal treatment is
administering both hMN-15 and DTIC for 3 days, and administering only
hMN-15 on days 7, 14, 21 and then every 21 days for a treatment duration
of 12 months. The doses of hMN-15 are 0.5-15 mg/kg body weight per
infusion, more preferably 2-8, and still more preferably 3-5 mg/kg per
infusion, and the doses of DTIC are as currently applied at the preferred
dose clinically, but could also be given at two-thirds or less of the
maximum preferred dose in use, thereby decreasing drug-related adverse
events. Repeated drug cycles can be given, such as every 1-6 months, with
continuation of the naked antibody therapy, or with different schedules
of radiolabeled antibody, drug-conjugated antibody, and inclusion of
certain cytokines, such as G-CSF and/or GM-CSF, each dose adjusted so
that toxicity to the patient is not enhanced by the therapeutic
combination. The application of a cytokine growth factor, such as G-CSF,
may enable even higher doses of myelosuppressive agents, such as
radiolabeled antibody or cytotoxic drugs, to be administered, and these
schedules and doses may be adjusted for the patients individually,
depending on their disease status and prior therapy, bone marrow status
and tolerability to additional cytotoxic therapies. In a preferred
embodiment, the Class I anti-CEA antibody or fragment thereof is
administered in a dosage of 100-600 milligrams protein per dose per
injection. Still more preferred, the Class I anti-CEA antibody or
fragment thereof is administered in a dosage of 300-400 milligrams of
protein per dose per injection, with repeated doses preferred. The
preferred antibody schedule is infusing once weekly or even less
frequently, such as once every other week or even every third week,
depending on a number of factors, including the extent of the disease and
the amount of CEA circulating in the patient's blood.

[0090] Bispecific and Multispecific Antibodies

[0091] In various embodiments, the Class I anti-CEA antibody comprising
light chain variable region CDR sequences SASSRVSYIH (SEQ ID NO:1);
GTSTLAS (SEQ ID NO:2); and QQWSYNPPT (SEQ ID NO:3); and heavy chain
variable region CDR sequences DYYMS (SEQ ID NO:4); FIANKANGHTTDYSPSVKG
(SEQ ID NO:5); and DMGIRWNFDV (SEQ ID NO:6) may be incorporated into a
bispecific or multispecific antibody. Bispecific antibodies are useful in
a number of biomedical applications. For instance, pre-targeting methods
with bispecific antibodies comprising at least one binding site for a
tumor-associated antigen (TAA), such as CEACAM 5 and/or CEACAM6, as well
as at least one binding site for a targetable construct conjugated to
therapeutic or diagnostic agents, are also well known in the art (see,
e.g., U.S. Pat. Nos. 7,300,644; 7,138,103; 7,074,405; 7,052,872;
6,962,702; 6,458,933, the Examples section of each of which is
incorporated herein by reference). In other embodiments, bispecific
antibodies comprising binding moieties targeting two different TAAs, or
different epitopes of the same TAA, may be of therapeutic use.

[0094] Numerous methods to produce bispecific or multispecific antibodies
are known, as disclosed, for example, in U.S. Pat. No. 7,405,320, the
Examples section of which is incorporated herein by reference. Bispecific
antibodies can be produced by the quadroma method, which involves the
fusion of two different hybridomas, each producing a monoclonal antibody
recognizing a different antigenic site (Milstein and Cuello, Nature,
1983; 305:537-540).

[0095] Another method for producing bispecific antibodies uses
heterobifunctional cross-linkers to chemically tether two different
monoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez,
et al. Nature. 1985; 316:354-356). Bispecific antibodies can also be
produced by reduction of each of two parental monoclonal antibodies to
the respective half molecules, which are then mixed and allowed to
reoxidize to obtain the hybrid structure (Staerz and Bevan. Proc Natl
Acad Sci USA. 1986; 83:1453-1457). Another alternative involves
chemically cross-linking two or three separately purified Fab' fragments
using appropriate linkers. (See, e.g., European Patent Application
0453082).

[0096] Other methods include improving the efficiency of generating hybrid
hybridomas by gene transfer of distinct selectable markers via
retrovirus-derived shuttle vectors into respective parental hybridomas,
which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.
1990, 87:2941-2945); or transfection of a hybridoma cell line with
expression plasmids containing the heavy and light chain genes of a
different antibody.

[0097] Cognate VH and VL domains can be joined with a peptide
linker of appropriate composition and length (usually consisting of more
than 12 amino acid residues) to form a single-chain Fv (scFv) with
binding activity. Methods of manufacturing scFvs are disclosed in U.S.
Pat. No. 4,946,778 and U.S. Pat. No. 5,132,405, the Examples section of
each of which is incorporated herein by reference. Reduction of the
peptide linker length to less than 12 amino acid residues prevents
pairing of VH and VL domains on the same chain and forces
pairing of VH and VL domains with complementary domains on
other chains, resulting in the formation of functional multimers.
Polypeptide chains of VH and VL domains that are joined with
linkers between 3 and 12 amino acid residues form predominantly dimers
(termed diabodies). With linkers between 0 and 2 amino acid residues,
trimers (termed triabodies) and tetramers (termed tetrabodies) are
favored, but the exact patterns of oligomerization appear to depend on
the composition as well as the orientation of V-domains
(VH-linker-VL or VL-linker-VH), in addition to the
linker length.

[0098] These techniques for producing multispecific or bispecific
antibodies exhibit various difficulties in terms of low yield, necessity
for purification, low stability or the labor-intensiveness of the
technique. More recently, a technique known as "dock and lock" (DNL) has
been utilized to produce combinations of virtually any desired
antibodies, antibody fragments and other effector molecules (see, e.g.,
U.S. Pat. Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400
and U.S. patent application Ser. Nos. 12/418,877; 12/544,476; 12/731,781;
12/752,649; and 12/754,740, the Examples section of each of which is
incorporated herein by reference).

[0099] Dock-and-Lock (DNL)

[0100] In preferred embodiments, bispecific or multispecific antibodies or
other constructs may be produced using the dock-and-lock technology. The
DNL method exploits specific protein/protein interactions that occur
between the regulatory (R) subunits of cAMP-dependent protein kinase
(PKA) and the anchoring domain (AD) of A-kinase anchoring proteins
(AKAPs) (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott,
Nat. Rev. Mol. Cell. Biol. 2004; 5: 959). PKA, which plays a central role
in one of the best studied signal transduction pathways triggered by the
binding of the second messenger cAMP to the R subunits, was first
isolated from rabbit skeletal muscle in 1968 (Walsh et al., J. Biol.
Chem. 1968; 243:3763). The structure of the holoenzyme consists of two
catalytic subunits held in an inactive form by the R subunits (Taylor, J.
Biol. Chem. 1989; 264:8443). Isozymes of PKA are found with two types of
R subunits (R1 and RID, and each type has α and β isoforms
(Scott, Pharmacol. Ther. 1991; 50:123). The R subunits have been isolated
only as stable dimers and the dimerization domain has been shown to
consist of the first 44 amino-terminal residues (Newlon et al., Nat.
Struct. Biol. 1999; 6:222). Binding of cAMP to the R subunits leads to
the release of active catalytic subunits for a broad spectrum of
serine/threonine kinase activities, which are oriented toward selected
substrates through the compartmentalization of PKA via its docking with
AKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561).

[0102] We have developed a platform technology to utilize the DDD of human
RIIα and the AD of AKAP as an excellent pair of linker modules for
docking any two entities, referred to hereafter as A and B, into a
noncovalent complex, which could be further locked into a stably tethered
structure through the introduction of cysteine residues into both the DDD
and AD at strategic positions to facilitate the formation of disulfide
bonds. The general methodology of the "dock-and-lock" approach is as
follows. Entity A is constructed by linking a DDD sequence to a precursor
of A, resulting in a first component hereafter referred to as a. Because
the DDD sequence would effect the spontaneous formation of a dimer, A
would thus be composed of a2. Entity B is constructed by linking an
AD sequence to a precursor of B, resulting in a second component
hereafter referred to as b. The dimeric motif of DDD contained in a2
will create a docking site for binding to the AD sequence contained in b,
thus facilitating a ready association of a2 and b to form a binary,
trimeric complex composed of a2b. This binding event is made
irreversible with a subsequent reaction to covalently secure the two
entities via disulfide bridges, which occurs very efficiently based on
the principle of effective local concentration because the initial
binding interactions should bring the reactive thiol groups placed onto
both the DDD and AD into proximity (Chmura et al., Proc. Natl. Acad. Sci.
USA. 2001; 98:8480) to ligate site-specifically. Using various
combinations of linkers, adaptor modules and precursors, a wide variety
of DNL constructs of different stoichiometry may be produced and used,
including but not limited to dimeric, trimeric, tetrameric, pentameric
and hexameric DNL constructs.

[0103] By attaching the DDD and AD away from the functional groups of the
two precursors, such site-specific ligations are also expected to
preserve the original activities of the two precursors. This approach is
modular in nature and potentially can be applied to link,
site-specifically and covalently, a wide range of substances, including
peptides, proteins, antibodies, antibody fragments, and other effector
moieties with a wide range of activities. Utilizing the fusion protein
method of constructing AD and DDD conjugated effectors described in the
Examples below, virtually any protein or peptide may be incorporated into
a DNL construct. However, the technique is not limiting and other methods
of conjugation may be utilized.

[0104] A variety of methods are known for making fusion proteins,
including nucleic acid synthesis, hybridization and/or amplification to
produce a synthetic double-stranded nucleic acid encoding a fusion
protein of interest. Such double-stranded nucleic acids may be inserted
into expression vectors for fusion protein production by standard
molecular biology techniques (see, e.g. Sambrook et al., Molecular
Cloning, A laboratory manual, 2nd Ed, 1989). In such preferred
embodiments, the AD and/or DDD moiety may be attached to either the
N-terminal or C-terminal end of an effector protein or peptide. However,
the skilled artisan will realize that the site of attachment of an AD or
DDD moiety to an effector moiety may vary, depending on the chemical
nature of the effector moiety and the part(s) of the effector moiety
involved in its physiological activity. Site-specific attachment of a
variety of effector moieties may be performed using techniques known in
the art, such as the use of bivalent cross-linking reagents and/or other
chemical conjugation techniques.

[0105] Pre-Targeting

[0106] Bispecific or multispecific antibodies may be utilized in
pre-targeting techniques. Pre-targeting is a multistep process originally
developed to resolve the slow blood clearance of directly targeting
antibodies, which contributes to undesirable toxicity to normal tissues
such as bone marrow. With pre-targeting, a radionuclide or other
therapeutic agent is attached to a small delivery molecule (targetable
construct or targetable conjugate) that is cleared within minutes from
the blood. A pre-targeting Bispecific or multispecific antibody, which
has binding sites for the targetable construct as well as a target
antigen, is administered first, free antibody is allowed to clear from
circulation and then the targetable construct is administered.

[0108] A pre-targeting method of treating or diagnosing a disease or
disorder in a subject may be provided by: (1) administering to the
subject a bispecific antibody or antibody fragment; (2) optionally
administering to the subject a clearing composition, and allowing the
composition to clear the antibody from circulation; and (3) administering
to the subject the targetable construct, containing one or more chelated
or chemically bound therapeutic or diagnostic agents. The technique may
also be utilized for antibody dependent enzyme prodrug therapy (ADEPT) by
administering an enzyme conjugated to a targetable construct, followed by
a prodrug that is converted into active form by the enzyme.

[0109] Therapeutic and Diagnostic Agents

[0110] In certain embodiments, the antibodies, antibody fragments or
fusion proteins described herein may be administered alone, as a "naked"
antibody, fragment or fusion protein. In alternative embodiments, the
antibody, fragment or fusion protein may be administered before,
concurrently with, or after at least one other therapeutic agent. In
other alternatives, an antibody, fragment or fusion protein may be
covalently or non-covalently attached to at least one therapeutic and/or
diagnostic agent to form an immunoconjugate.

[0111] Diagnostic agents are preferably selected from the group consisting
of a radionuclide, a radiological contrast agent, a paramagnetic ion, a
metal, a fluorescent label, a chemiluminescent label, an ultrasound
contrast agent and a photoactive agent. Such diagnostic agents are well
known and any such known diagnostic agent may be used. Non-limiting
examples of diagnostic agents may include a radionuclide such as
110In, 111In, 177Lu, 18F, 52Fe, 62Cu,
64Cu, 67Cu, 67Ga, 68Ga, 86Y, 90Y,
89Zr, 94mTc, 94Tc, 99mTc, 120I, 123I,
124I, 125I, 131I, 154-158Gd, 32P, 11C,
13N, 15O, 186Re, 188Re, 51Mn, 52mMn,
55Co, 72As, 75Br, 76Br, 82mRb, 83Sr, or
other gamma-, beta-, or positron-emitters. Paramagnetic ions of use may
include chromium (III), manganese (II), iron (III), iron (II), cobalt
(II), nickel (II), copper (II), neodymium (III), samarium (III),
ytterbium (III), gadolinium (III), vanadium (II), terbium (III),
dysprosium (III), holmium (III) or erbium (III). Metal contrast agents
may include lanthanum (III), gold (III), lead (II) or bismuth (III).
Ultrasound contrast agents may comprise liposomes, such as gas filled
liposomes. Radiopaque diagnostic agents may be selected from barium
compounds, gallium compounds and thallium compounds. A wide variety of
fluorescent labels are known in the art, including but not limited to
fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin,
allophycocyanin, o-phthaldehyde and fluorescamine. Chemiluminescent
labels of use may include luminol, isoluminol, an aromatic acridinium
ester, an imidazole, an acridinium salt or an oxalate ester.

[0112] Therapeutic agents are preferably selected from the group
consisting of a radionuclide, an immunomodulator, an anti-angiogenic
agent, a cytokine, a chemokine, a growth factor, a hormone, a drug, a
prodrug, an enzyme, an oligonucleotide, a pro-apoptotic agent, a
photoactive therapeutic agent, a cytotoxic agent, which may be a
chemotherapeutic agent or a toxin, and a combination thereof. The drugs
of use may possess a pharmaceutical property selected from the group
consisting of antimitotic, antikinase, alkylating, antimetabolite,
antibiotic, alkaloid, anti-angiogenic, pro-apoptotic agents and
combinations thereof.

[0119] Corticosteroid hormones can increase the effectiveness of other
chemotherapy agents, and consequently, they are frequently used in
combination treatments. Prednisone and dexamethasone are examples of
corticosteroid hormones.

[0121] Other useful therapeutic agents comprise oligonucleotides,
especially antisense oligonucleotides that preferably are directed
against oncogenes and oncogene products of B-cell malignancies, such as
bcl-2. Preferred antisense oligonucleotides include those known as siRNA
or RNAi.

[0122] Immunoconjugates

[0123] Any of the antibodies, antibody fragments or antibody fusion
proteins described herein may be conjugated to one or more therapeutic or
diagnostic agents. The therapeutic agents do not need to be the same but
can be different, e.g. a drug and a radioisotope. For example, 1311
can be incorporated into a tyrosine of an antibody or fusion protein and
a drug attached to an epsilon amino group of a lysine residue.
Therapeutic and diagnostic agents also can be attached, for example to
reduced SH groups and/or to carbohydrate side chains. Many methods for
making covalent or non-covalent conjugates of therapeutic or diagnostic
agents with antibodies or fusion proteins are known in the art and any
such known method may be utilized.

[0124] A therapeutic or diagnostic agent can be attached at the hinge
region of a reduced antibody component via disulfide bond formation.
Alternatively, such agents can be attached using a heterobifunctional
cross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). Yu
et al., Int. J. Cancer 56: 244 (1994). General techniques for such
conjugation are well-known in the art. See, for example, Wong, CHEMISTRY
OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et
al., "Modification of Antibodies by Chemical Methods," in MONOCLONAL
ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages
187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization
of Synthetic Peptide-Derived Antibodies," in MONOCLONAL ANTIBODIES:
PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),
pages 60-84 (Cambridge University Press 1995). Alternatively, the
therapeutic or diagnostic agent can be conjugated via a carbohydrate
moiety in the Fc region of the antibody. The carbohydrate group can be
used to increase the loading of the same agent that is bound to a thiol
group, or the carbohydrate moiety can be used to bind a different
therapeutic or diagnostic agent.

[0125] Methods for conjugating peptides to antibody components via an
antibody carbohydrate moiety are well-known to those of skill in the art.
See, for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et
al., Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.
5,057,313, incorporated herein by reference. The general method involves
reacting an antibody component having an oxidized carbohydrate portion
with a carrier polymer that has at least one free amine function. This
reaction results in an initial Schiff base (imine) linkage, which can be
stabilized by reduction to a secondary amine to form the final conjugate.

[0126] The Fc region may be absent if the antibody used as the antibody
component of the immunoconjugate is an antibody fragment. However, it is
possible to introduce a carbohydrate moiety into the light chain variable
region of a full length antibody or antibody fragment. See, for example,
Leung et al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S. Pat. No.
5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868, each
incorporated herein by reference. The engineered carbohydrate moiety is
used to attach the therapeutic or diagnostic agent.

[0127] In some embodiments, a chelating agent may be attached to an
antibody, antibody fragment or fusion protein or to a targetable
construct and used to chelate a therapeutic or diagnostic agent, such as
a radionuclide. Exemplary chelators include but are not limited to DTPA
(such as Mx-DTPA), DOTA, TETA, NETA or NOTA. Methods of conjugation and
use of chelating agents to attach metals or other ligands to proteins are
well known in the art (see, e.g., U.S. Pat. No. 7,563,433, the Examples
section of which is incorporated herein by reference).

[0128] In certain embodiments, radioactive metals or paramagnetic ions may
be attached to proteins or peptides by reaction with a reagent having a
long tail, to which may be attached a multiplicity of chelating groups
for binding ions. Such a tail can be a polymer such as a polylysine,
polysaccharide, or other derivatized or derivatizable chains having
pendant groups to which can be bound chelating groups such as, e.g.,
ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic
acid (DTPA), porphyrins, polyamines, crown ethers,
bis-thiosemicarbazones, polyoximes, and like groups known to be useful
for this purpose.

[0129] Chelates may be directly linked to antibodies or peptides, for
example as disclosed in U.S. Pat. No. 4,824,659, incorporated herein by
reference. Particularly useful metal-chelate combinations include
2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with
diagnostic isotopes in the general energy range of 60 to 4,000 keV, such
as 125I, 131I, 123I, 124I, 62Cu, 64Cu,
18F, 111In, 67Ga, 68Ga, 99mTc, 94mTc,
11C, 13N, 15O, 76Br, for radio-imaging. The same
chelates, when complexed with non-radioactive metals, such as manganese,
iron and gadolinium are useful for MRI. Macrocyclic chelates such as
NOTA, DOTA, and TETA are of use with a variety of metals and radiometals,
most particularly with radionuclides of gallium, yttrium and copper,
respectively. Such metal-chelate complexes can be made very stable by
tailoring the ring size to the metal of interest. Other ring-type
chelates such as macrocyclic polyethers, which are of interest for stably
binding nuclides, such as 223Ra for RAIT are encompassed.

[0130] More recently, methods of 18F-labeling of use in PET scanning
techniques have been disclosed, for example by reaction of F-18 with a
metal or other atom, such as aluminum. The 18F--Al conjugate may be
complexed with chelating groups, such as DOTA, NOTA or NETA that are
attached directly to antibodies or used to label targetable constructs in
pre-targeting methods. Such F-18 labeling techniques are disclosed in
U.S. Pat. No. 7,563,433, the Examples section of which is incorporated
herein by reference.

[0131] Pharmaceutically Acceptable Vehicles

[0132] The compositions comprising murine, humanized, chimeric or human
Class I anti-CEA MAbs to be delivered to a subject can comprise one or
more pharmaceutically acceptable vehicles, one or more additional
ingredients, or some combination of these. The Class I anti-CEA
antibodies and fragments thereof can be formulated according to known
methods to prepare pharmaceutically useful compositions. Sterile
phosphate-buffered saline is one example of a pharmaceutically acceptable
vehicle. Other acceptable vehicles are well-known to those in the art.
See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG
DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),
REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing
Company 1990), and revised editions thereof.

[0133] The Class I anti-CEA antibody or fragment thereof can be formulated
for intravenous administration via, for example, bolus injection or
continuous infusion. Formulations for injection can be presented in unit
dosage form, e.g., in ampules or in multi-dose containers, with an added
preservative. The compositions can take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and can contain
formulatory agents such as suspending, stabilizing and/or dispersing
agents. Alternatively, the active ingredient can be in powder form for
reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,
before use.

[0134] Additional pharmaceutical methods may be employed to control the
duration of action of the therapeutic agent and/or antibody or fragment
thereof. Control release preparations can be prepared through the use of
polymers to complex or adsorb the antibody. For example, biocompatible
polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices
of a polyanhydride copolymer of a stearic acid dimer and sebacic acid.
Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release of
an antibody or fragment thereof from such a matrix depends upon the
molecular weight of the immunoconjugate or antibody, the amount of
antibody within the matrix, and the size of dispersed particles. Saltzman
et al., Biophys. J. 55: 163 (1989); Sherwood et al., supra. Other solid
dosage forms are described in Ansel et al., PHARMACEUTICAL DOSAGE FORMS
AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro
(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing
Company 1990), and revised editions thereof.

[0135] The Class I anti-CEA antibody or fragment thereof may also be
administered to a mammal subcutaneously or by other parenteral routes.
Moreover, the administration may be by continuous infusion or by single
or multiple boluses. In general, the dosage of an administered antibody
or fragment thereof for humans will vary depending upon such factors as
the patient's age, weight, height, sex, general medical condition and
previous medical history. Typically, it is desirable to provide the
recipient with a dosage of antibody or fragment thereof that is in the
range of from about 0.5 mg/kg to 20 mg/kg as a single intravenous
infusion, although a lower or higher dosage also may be administered as
circumstances dictate. This dosage may be repeated as needed, for
example, once per month for 4-10 months, preferably once per every other
week for 16 weeks, and more preferably, once per week for 8 weeks. It may
also be given less frequently, such as every other week for several
months or given more frequently and/or over a longer duration. The dosage
may be given through various parenteral routes, with appropriate
adjustment of the dose and schedule.

[0136] For purposes of therapy, the Class I anti-CEA antibody or fragment
thereof is administered to a mammal in a therapeutically effective amount
to reduce the size of a tumor as compared to untreated controls.
Preferably, the Class I anti CEA antibody or fragment thereof is a
humanized antibody or fragment thereof. A suitable subject for the
present invention is usually a human, although a non-human mammal or
animal subject is also contemplated. An antibody preparation is
administered in a "therapeutically effective amount" if the amount
administered is physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient mammal.

[0137] In particular, an antibody preparation is physiologically
significant if its presence invokes an antitumor response. A
physiologically significant effect could also be the evocation of a
humoral and/or cellular immune response in the recipient mammal.

[0138] Kits

[0139] Various embodiments may concern kits containing components suitable
for treating or diagnosing diseased tissue in a patient. Exemplary kits
may contain at least one antibody, antibody fragment or fusion protein as
described herein. If the composition containing components for
administration is not formulated for delivery via the alimentary canal,
such as by oral delivery, a device capable of delivering the kit
components through some other route may be included. One type of device,
for applications such as parenteral delivery, is a syringe that is used
to inject the composition into the body of a subject. Inhalation devices
may also be used. In certain embodiments, an Class I anti-CEA antibody or
fragment thereof may be provided in the form of a prefilled syringe or
autoinjection pen containing a sterile, liquid formulation or lyophilized
preparation of antibody (e.g., Kivitz et al., Clin. Ther. 2006,
28:1619-29).

[0140] The kit components may be packaged together or separated into two
or more containers. In some embodiments, the containers may be vials that
contain sterile, lyophilized formulations of a composition that are
suitable for reconstitution. A kit may also contain one or more buffers
suitable for reconstitution and/or dilution of other reagents. Other
containers that may be used include, but are not limited to, a pouch,
tray, box, tube, or the like. Kit components may be packaged and
maintained sterilely within the containers. Another component that can be
included is instructions to a person for use of the kit.

[0143] Various embodiments relate to expression vectors comprising the
coding DNA sequences. The vectors may contain sequences encoding the
light and heavy chain constant regions and the hinge region of a human
immunoglobulin to which may be attached chimeric, humanized or human
variable region sequences. The vectors may additionally contain promoters
that express MAbs in a selected host cell, immunoglobulin enhancers and
signal or leader sequences. Vectors that are particularly useful are
pdHL2 or GS. More preferably, the light and heavy chain constant regions
and hinge region may be from a human EU myeloma immunoglobulin, where
optionally at least one of the amino acid in the allotype positions is
changed to that found in a different IgG1 allotype, and wherein
optionally amino acid 253 of the heavy chain of EU based on the EU number
system may be replaced with alanine. See Edelman et al., Proc. Natl.
Acad. Sci. USA 63: 78-85 (1969).

[0144] Also encompassed is a method of expressing antibodies or fragments
thereof or fusion proteins. The skilled artisan will realize that methods
of genetically engineering expression constructs and insertion into host
cells to express engineered proteins are well known in the art and a
matter of routine experimentation. Host cells and methods of expression
of cloned antibodies or fragments have been described, for example, in
U.S. Pat. Nos. 7,531,327; 7,537,930 and 7,608,425, the Examples section
of each of which is incorporated herein by reference.

[0145] General Techniques for Construction of Anti-CEA Antibodies

[0146] The V.sub.κ (variable light chain) and VH (variable
heavy chain) sequences for Class I anti-CEA antibodies may be obtained by
a variety of molecular cloning procedures, such as RT-PCR, 5'-RACE, and
cDNA library screening. Specifically, the V genes of a Class I anti-CEA
MAb from a cell that expresses a murine Class I anti-CEA MAb can be
identified by PCR amplification and DNA sequencing. To confirm their
authenticity, the cloned VL and VH genes can be expressed in
cell culture as a chimeric Ab as described by Orlandi et al., (Proc.
Natl. Acad. Sci., USA, 86: 3833 (1989)). Based on the V gene sequences, a
humanized Class I anti-CEA MAb can then be designed and constructed as
described by Leung et al. (Mol. Immunol., 32: 1413 (1995)).

[0147] cDNA can be prepared from any known hybridoma line or transfected
cell line producing a murine Class I anti-CEA MAb by general molecular
cloning techniques (Sambrook et al., Molecular Cloning, A laboratory
manual, 2nd Ed (1989)). The V.sub.κ sequence for the MAb may
be amplified using the primers VK1BACK and VK1FOR (Orlandi et al., 1989)
or the extended primer set described by Leung et al. (BioTechniques, 15:
286 (1993)). The VH sequences can be amplified using the primer pair
VH1BACK/VH1FOR (Orlandi et al., 1989) or the primers annealing to the
constant region of murine IgG described by Leung et al. (Hybridoma,
13:469 (1994)).

[0149] PCR products for V.sub.κ can be subcloned into a staging
vector, such as a pBR327-based staging vector, VKpBR, that contains an Ig
promoter, a signal peptide sequence and convenient restriction sites to
facilitate in-frame ligation of the V.sub.κ PCR products. PCR
products for VH can be subcloned into a similar staging vector, such
as the pBluescript-based VHpBS. Individual clones containing the
respective PCR products may be sequenced by, for example, the method of
Sanger et al. (Proc. Natl. Acad. Sci., USA, 74: 5463 (1977)).

[0150] Expression cassettes containing the V.sub.κ and VH
sequences, together with the promoter and signal peptide sequences, can
be excised from VKpBR and VHpBS, respectively, by double restriction
digestion as HindIII-BamHI fragments. The V.sub.κ and VH
expression cassettes can be ligated into appropriate expression vectors,
such as pKh and pG1g, respectively (Leung et al., Hybridoma, 13:469
(1994)). The expression vectors can be co-transfected into an appropriate
cell, e.g., myeloma Sp2/0-Ag14 (ATCC, VA), colonies selected for
hygromycin resistance, and supernatant fluids monitored for production of
a chimeric, humanized or human. Class I anti-CEA MAb by, for example, an
ELISA assay. Alternatively, the V.sub.κ and VH expression
cassettes can be assembled in the modified staging vectors, VKpBR2 and
VHpBS2, excised as XbaI/BamHI and XhoI/BamHI fragments, respectively, and
subcloned into a single expression vector, such as pdHL2, as described by
Gilles et al. (J. Immunol. Methods 125:191 (1989) and also shown in
Losman et al., Cancer, 80:2660 (1997)). Another vector that is useful is
the GS vector, as described in Barnes et al., Cytotechnology 32:109-123
(2000). Other appropriate mammalian expression systems are described in
Werner et al., Arzneim.-Forsch./Drug Res. 48(II), Nr. 8, 870-880 (1998).

[0151] Co-transfection and assay for antibody secreting clones by ELISA
can be carried out as follows. About 10 μg of VKpKh (light chain
expression vector) and 20 μg of VHpG1g (heavy chain expression vector)
can be used for the transfection of 5×106 SP2/0 myeloma cells
by electroporation (BioRad, Richmond, Calif.) according to Co et al., J.
Immunol., 148: 1149 (1992). Following transfection, cells may be grown in
96-well microliter plates in complete HSFM medium (Life Technologies,
Inc., Grand Island, N.Y.) at 37° C., 5% CO2. The selection
process can be initiated after two days by the addition of hygromycin
selection medium (Calbiochem, San Diego, Calif.) at a final concentration
of 500 units/ml of hygromycin. Colonies typically emerge 2-3 weeks
post-electroporation. The cultures can then be expanded for further
analysis. Transfectoma clones that are positive for the secretion of
chimeric, humanized or human heavy chain can be identified by ELISA
assay.

[0152] Antibodies can be isolated from cell culture media as follows.
Transfectoma cultures are adapted to serum-free medium. For production of
chimeric antibody, cells are grown as a 500 ml culture in roller bottles
using HSFM. Cultures are centrifuged and the supernatant filtered through
a 0.2μ membrane. The filtered medium is passed through a protein A
column (1×3 cm) at a flow rate of 1 ml/min. The resin is then
washed with about 10 column volumes of PBS and protein A-bound antibody
is eluted from the column with 0.1 M glycine buffer (pH 3.5) containing
10 mM EDTA. Fractions of 1.0 ml are collected in tubes containing 10
μl of 3 M Tris (pH 8.6), and protein concentrations determined from
the absorbance at 280/260 nm. Peak fractions are pooled, dialyzed against
PBS, and the antibody concentrated, for example, with the Centricon 30
(Amicon, Beverly, Mass.). The antibody concentration is determined by
ELISA and its concentration adjusted to about 1 mg/ml using PBS. Sodium
azide, 0.01% (w/v), is conveniently added to the sample as preservative.

EXAMPLES

[0153] The invention is further illustrated by, though in no way limited
to, the following examples.

Example 1

Production and Characterization of MN-15 and Other Anti-CEA Antibodies

[0154] In 1983, Primus et al. described the first panel of MAbs (NP-1,
NP-2, NP-3, and NP-4) that defined NCA-cross-reactive, MA-cross-reactive,
and CEA-specific epitopes on the CEA molecule (Primus et al., Cancer Res
1983, 43:686-92). NP-1 reacts with NCA (normal cross-reactive antigen),
MA (meconium antigen), and CEA (carcinoembryonic antigen) and, in a
liquid solution of decreasing ion strength, demonstrates increasing
affinity for CEA, a property shared with unadsorbed goat anti-CEA serum
and affinity-purified NCA crossreactive antibody purified from goat
anti-CEA serum (Primus et al., 1983). The "ion-sensitive" determinant on
CEA first was delineated by Hansen et al. (Clin Res 1971, 19:143) using
unadsorbed anti-CEA polyclonal serum, and subsequently by Haskell et al.
(Cancer Res 1983, 43:3857-64) with MAb. NP-2 and NP-3 react with MA and
CEA but not with NCA, whereas NP-4 reacts only with CEA (Primus et al.,
1983). NP-2 and NP-3 differ because NP-2 binding to CEA is blocked by
NP-1, whereas NP-1 does not block binding of NP-3 to CEA. MAb reactive
with NCA, MA, and CEA were designated as Class I MAb; MAb reactive with
MA and CEA but unreactive with NCA were designated as Class II MAb; and
MAb specific for CEA, being unreactive with NCA and MA, were designated
as Class III MAb (see, e.g., U.S. Pat. No. 4,818,709, the Examples
section of which is incorporated herein by reference). The present
Example describes the production and characterization of a second
generation of anti-CEA antibodies, including the MN-15 Class I anti-CEA
MAb.

[0155] Immunization and Hybridoma Production and Screening

[0156] The immunogen used to produce MN-15 and other second generation
anti-CEA antibodies was a partially purified CEA preparation derived from
the GW-39 human colon adenocarcinoma xenograft (Hansen et al., 1993,
Cancer 71:3478-85). Tumor was grown in the hind-leg muscle of the golden
hamster, excised, frozen, and stored at -20° C. Homogenization of
tumor, extraction with perchloric acid, and concentration of the dialyzed
extract by ultrafiltration were performed as described previously (Newman
et al., Cancer Res 1974, 34:2125-530). The concentrated extract was
equilibrated by dialysis with saline adjusted to 0.05 M
NaH2PO4, and chromatographed on a 5.0×90.0 cm G-200
SEPHADEX® gel column. The void peak was collected, equilibrated with
saline by dialysis, and frozen at -20° C. until used. The
concentration of CEA in the extract was determined with the
IMMUNOMEDICS® NP-1/NP-3 and NP-1/NP-4 enzyme immunoassays (EIA)
(Immunomedics, Inc., Morris Plains, N.J.).

[0157] The following protocol was used to immunize 20-g BALB/c female mice
(Harlan, Madison, Wis.), from which the second-generation MN series of
anti-CEA MAb were derived. Mice first were immunized subcutaneously with
7.5 μg of CEA in complete Freund adjuvant and then boosted
subcutaneously with 7.5 μg of CEA in incomplete Freund adjuvant on day
3 and boosted intravenously with 7.5 μg of CEA in saline on days 6 and
9. On day 20, the first mouse was given 50 μg of CEA intravenously in
saline; on day 23 one animal was killed, the spleen cell suspensions were
prepared, and the cells were fused with murine myeloma cells SP2/0-Ag 14
with the use of polyethylene glycol and then cultured in medium
containing 8-azaguanine.

[0158] Hybridoma supernatants were screened for CEA reactive antibody by
the ROCHE® 125I-CEA radioimmunoassay (RIA) in 0.01 and 0.10 M
NH4 acetate buffer. Positive clones were recloned. The granulocyte
reactivity of CEA-reactive MAb was determined by whole-blood indirect
fluorescent flow cytometric analysis (Sharkey et al. Cancer Res., 1990,
50:2823-31). Two MAb with properties similar to those of NP-2 and NP-3,
designated MN-2 and MN-6, were selected for additional development. A
third MAb, MN-3, reacted with granulocytes but, unlike NP-1 and NP-2, did
not demonstrate ion sensitivity for CEA binding; it also was selected for
development.

[0159] The mouse used in the second fusion was immunized on days 1, 3, 6,
and 9, as described above. It was given 65 μg of CEA in saline
intravenously on day 278 and 90 μg of CEA in saline on day 404. It was
killed on day 407. The fusion and screening protocols were the same as
those used for the first mouse. The second fusion yielded one clone
(MN-14) that had properties consistent with those of NP-4 in that it was
unreactive with granulocytes, bound only 30-40% of 125I-CEA in the
ROCHE® CEA RIA, and demonstrated no ion sensitivity of CEA binding in
the RIA. A second clone producing a granulocyte-reactive antibody with
properties identical to those of NP-1, MN-15, also was derived from this
fusion.

[0160] MAb-IgG Purification/Characterization

[0161] The MAb were purified from ascites fluid raised in CD2F1 mice
(Charles River Laboratories, Wilmington, Mass.) by protein A and
ion-exchange column chromatography at 4° C. All of the MAb were of
the IgG1 isotype with kappa light chains, as determined by double gel
diffusion.

[0162] Enzyme Immunoassay

[0163] The reagents, formulation, standards, and assay protocol of the
sandwich EIA used in these studies have been described elsewhere (Hansen
et al., Clin Chem 1989, 35:146-51). All assays were performed with
microwells coated with NP-1, except in one experiment, in which
microwells were coated with MN-15. All assays were performed in a
sequential mode, with addition of CEA first, incubation, washing of the
wells, and addition of the MAb-horseradish peroxidase probe. After the
second incubation, the amount of probe bound to CEA was determined by
washing out the unbound probe, adding substrate
(o-phenylenediamine/hydrogen peroxide), adding acid to stop the reaction,
and quantitating the color product by reading the absorbance at 490 nm.
Horseradish peroxidase was conjugated to MAb NP-4, MN-3, MN-6, and MN-14,
as described previously for NP-3 (Hansen et al., Clin Chem 1989,
35:146-51). All conjugates were diluted to match the dose-response curve
established previously with the NP-3-horseradish peroxidase probe. Probes
made with NP-4, MN-3, MN-6, and MN-14 all gave linear dose-response
curves with the GW-39 CEA standard.

[0164] MA Standard

[0165] Meconium was homogenized in 10 volumes of deionized water and the
mixture centrifuged at 40,000×g for 30 minutes. The supernatant was
decanted from the pellet and stored at -20° C. The amount of MA
plus CEA (in ng/ml) in the MA standard was determined by assay with the
NP-1/NP-3 EIA. The amount of CEA (in ng/ml) in the MA standard was
determined with the NP-1/NP-4 assay and found to be 15% of the total
MA/CEA activity determined with the NP-1/NP-3 EIA.

[0166] Blocking Studies

[0167] Blocking studies were performed with the same conditions described
for quantification of CEA. To assess blocking of binding of the enzyme
probes, microwells were charged with CEA by use of 25 ng/ml of the CEA
standard. Unlabeled MAb were added to MAb-enzyme conjugates, and the EIA
assay was completed as described for quantitation of CEA with the NP-3
probe.

[0168] Tissue Reactivity

[0169] Immunoreactivity with blood lymphocytes was determined by live-cell
indirect immunofluorescence, with the use of the whole blood staining
method developed for use with the ORTHO SPECTRUM® I11 flow cytometer
(Ortho Diagnostic Systems, Inc., Raritan, N.J.). Tissue reactivity of the
MAb was determined by immunohistologic examination performed on frozen
sections and 5 μm sections cut from tissue embedded in Paraplast-II.
Indirect immunofluorescence on frozen sections was performed as
previously described, with MAb concentrations of 20 μg/ml
(Pawlak-Byczkowska et al., Cancer Res 1989, 49:4568-77) Immunoperoxidase
staining of sections was performed with the avidin-biotin horseradish
peroxidase complex method (Vector Laboratories, Burlingame, Calif.)
according to the protocol suggested by the manufacturer, with MAb
concentrations of 10 μg/ml.

[0170] Animal Studies

[0171] For radiolocalization studies, at 4-5 weeks female athymic mice
(nu/nu; Harlan, Indianapolis, Ind.) were given subcutaneous injections of
0.2 ml of a 10% suspension of GW-39, prepared from the tumor serially
propagated in athymic mice. After 2 weeks, the mice were given
intravenous injections of approximately 1 μg of 131I-labeled NP-4
or MN-14. The animals were killed 7 and 14 days later; the organs were
removed and radioactivity in the organs determined as described
previously (Sharkey et al., Cancer Res 1990, 50:828s-34s).

[0172] Results

[0173] MAb MN-3 and MN-6 reacted with MA, whereas MN-14 did not detect MA.
All of the MAb-horseradish peroxidase probes were titrated with
NP-1-coated microwells charged with 25 ng/ml of CEA to obtain an
absorbance of 0.6-0.8. Microwells coated with MN-15 yielded results
identical to those from microwells coated with NP-1. CEA and MA standards
were sent as coded specimens to commercial laboratories performing the
respective commercial assays. It was expected that the ROCHE® CEA EIA
would be specific for CEA because the ROCHE® assay uses MAb T86.44, a
MAb that reacts with CEA but not with MA (Neumaier et al., J Immunol
1985, 135:3604-09). Both the HYBRITECH® and ABBOTT® commercial
CEA EIA demonstrated cross-reactivity with MA (not shown).

[0174] The affinity of radioiodinated MN-14 for CEA was 10 times that of
NP-4 as determined by adding increasing amounts of CEA to fixed amounts
of MAb and determining free versus bound antibody by high pressure liquid
chromatography sizing gel analysis. The 100 ng/ml of radioactive antibody
used in this study to determine the affinity of the MAb approximates the
serum concentration of antibody present in blood after infusion of 1
μg of antibody. Between 1 and 2 μg/ml of CEA was required to
complex 50% of NP-4, with 50% complexation of MN-14 being obtained with
approximately 0.2 μg/ml of CEA (results not shown).

[0175] The normal tissue reactivity of NP-4 and MN-14 was found to be
identical, with both being unreactive with blood granulocytes, spleen,
normal liver, and all other normal tissues except the colon (not shown).
Staining of normal colon with both MAb was localized to the glycocalyx of
the epithelial cells (not shown). Staining of sections of colorectal
carcinoma from 10 different patients and of one lung carcinoma
demonstrated identical staining patterns with the two MAb, but more
intense staining was observed consistently with MN-14 (not shown).

[0176] Results of immunoperoxidase staining of spleen sections with MN-2
and MN-3, performed on frozen sections and sections prepared by
conventional tissue processing (fixed in formaldehyde solution and
embedded in Paraplast) were determined. The granulocyte antigen reactive
with MN-3 (spleen sections) was stained in sections fixed in formaldehyde
solution and embedded in Paraplast, whereas the antigen reactive with
MN-2 was not detected in granulocytes in the section fixed in
formaldehyde solution and embedded in Paraplast (not shown). By contrast,
MN-2 strongly stained CEA present in the section of colon carcinoma fixed
in formaldehyde solution and embedded in Paraplast (not shown). The
pattern of MN-2 staining was similar to that observed with MN-3 and
MN-14. Although blocking studies cannot prove that two MAb are reactive
with the same epitope, failure to bock provides evidence of reactivity
with separate epitopes.

[0177] To evaluate cross-blocking of NP-4 by MN-14, microwells coated with
NP-1 were incubated with 25 μg of CEA, with reagents described above.
The microwells then were washed and the NP-4-horseradish peroxidase probe
added, with and without 50 μg/ml of MN-14. After incubation, the
supernatants in the wells were decanted, the wells were washed, and the
assay was completed. Binding of the NP-4 probe was inhibited by greater
than 50%. No blocking of binding of the NP-3 probe was demonstrated under
similar experimental conditions. Blocking of binding of the NP-3 probe
was evaluated with the same protocol used above for NP-4. Both MN-3 and
MN-6 blocked binding of the NP-3 probe to CEA.

[0178] NP-1 and MN-15 appeared to have similar properties, demonstrating
high granulocyte reactivity, binding to epitopes of the ion-sensitive CEA
determinant, and rapidly effecting capture of CEA from solution when
coated on microwells (not shown). NP-2 and MN-2 bind to the ion-sensitive
determinant but differ from NP-1 in having much lower reactivity with
blood granulocytes and reacting with a granulocyte antigen that was
destroyed by conventional tissue processing used in histologic
examination (not shown). MN-3 reacted strongly with granulocytes but did
not react with the ion-sensitive determinant (not shown). It also reacted
with CEA bound to NP-1, whereas NP-2 demonstrated cross-blocking by NP-1
(not shown). NP-3, MN-6, NP-4, and MN-14 were not reactive with
granulocytes or the ion sensitive determinant (not shown). NP-3 and MN-6
reacted with MA, whereas NP-4 and MN-14 were not reactive with MA (not
shown).

[0179] Several tumor localization studies have been performed to compare
the effectiveness of radioiodinated NP-4 with that of MN-14, with
superior targeting being demonstrated for MN-14 in two studies and
equivalent targeting demonstrated in a third study (not shown). Twenty
animals were injected for each time point for each of the antibodies. As
was observed, MN-14, as compared with NP-4, provided significantly
improved tumor uptake at both 7 and 14 days after injection (P<0.02
and P<0.001, respectively). The uptake of the two antibodies in normal
organs or blood was not significantly different (not shown).

[0180] Discussion

[0181] MN-14 meets all of the criteria of a Class III anti-CEA MAb, being
unreactive with MA by EIA and not staining normal tissues, with the
exception of normal colon. This study adds support to the suggestion that
a lack of reactivity with MA is the single most useful parameter for
selecting Class III anti-CEA MAb. Blocking experiments alone can be
misleading in the classification of CEA MAb, as is apparent from the
extensive data published from the International Workshop on epitope
reactivity of MAb reactive with CEA (Hammarstrom et al., Cancer Res 1989,
49:4852-58). Although blocking experiments placed a number of Class III
MAb into the Gold Group 1 (T84.66, II-16, 11-7, II-10), CEA 66, which is
strongly reactive with MA and reacts weakly with NCA, cross-blocked CEA
binding of a Class III MAb, 11-16. MAb B7.8.5, also placed in Gold Group
1, blocked binding of 11-16 yet reacts with NCA. Thus, the Gold Group 1,
constructed on the basis of cross-blocking of binding of MAb to CEA,
contains Class I, II, and III MAb.

[0182] The affinity of MN-14 is approximately 10-fold greater than that of
NP-4, when determined with radioiodinated MAb and identical assay
conditions to determine free versus antigen-bound antibody. The affinity
determined for NP-4 in this study was significantly lower than reported
previously with 125I-CEA in place of radiolabeled NP-4 (Primus et
al., 1983). MN-14, like NP-4 and T86.44, binds less than 50% of
radioiodinated CEA. In the case of T86.44, the low binding of 125I
has been ascribed to damage of the T86.44-reactive epitope by
radioiodination. If radioiodination results in damage to the
NP-4-reactive epitope, unlabeled CEA would displace NP-4 more
effectively, resulting in a falsely elevated affinity constant. For this
reason, we elected to compare the affinity of NP-4 and MN-14 by a direct
binding method using radioiodinated antibody, rather than by competitive
blocking of binding of 125I-CEA to antibody.

[0183] This study also confirms the findings of Bormer et al. (Clin Chem
1991, 37:1736-39), that the T84.66 MAb, used as a probe in the commercial
ROCHE® CEA EIA, is unreactive with MA, whereas commercial ABBOTT®
and HYBRITECH® CEA EIA use MAb that react equally with MA and CEA.
Experiments in athymic mice bearing human colon cancer xenografts have
demonstrated consistently improved targeting with MN-14 compared with
NP-4 (not shown). The results of targeting with NP-4 are highly variable
between experiments, however; and it has not been possible to conclude
whether the superior targeting resulted from increased binding or longer
retention of the antibody in the tumor, as suggested by the data
presented herein.

[0184] Four of the MN MAb have been evaluated by clinical
radioscintigraphy studies. MN-3 strongly targets bone marrow granulocytes
and is being investigated for radioimmunodetection of occult infection
and inflammation. MN-2 and MN-6 demonstrated similar deficiencies in
radioimmunodetection of CEA-containing cancers, as observed for NP-2 and
NP-3. 131I-MN-2 targeted bone marrow, and 131I-MN-6 accreted in
the lumen of the normal colon (unpublished results). Excellent
specificity and sensitivity were demonstrated with 131I-MN-14 in a
Phase I clinical study to detect CEA containing tumors by
radioimmunodetection, and results of this study have been described
(Sharkey et al., Cancer 1993, 71:2082-96).

[0185] Conclusions

[0186] A second-generation panel of anti-carcinoembryonic antigen
(anti-CEA) monoclonal antibodies (MAb) has been generated, and the
specificity has been compared with that of the first panel of MAb used to
differentiate meconium antigen (MA) from CEA.

[0187] Four of the MAb had similar specificities to the first-generation
panel of NP MAb. MN-15, like its first-generation equivalent, NP-1,
reacted with normal cross-reactive antigen (NCA), MA, and CEA. Both MN-15
and NP-1 reacted strongly with granulocytes. MN-2 had properties similar
to Class II NP-2, being reactive with MA and CEA, cross-blocking binding
to CEA by NP-1, and having low reactivity with granulocytes. Both NP-2
and MN-2 stained granulocytes in frozen tissue sections but showed
minimal staining of granulocytes in sections fixed in formaldehyde
solution and embedded in Paraplast. MN-14 demonstrated properties similar
to the Class III anti-CEA-specific MAb, NP-4, being unreactive with NCA
and MA. MN-14, as compared with NP-4, demonstrated significantly superior
tumor targeting in a human colon tumor xenograft model and consistently
stronger staining of frozen sections of colon cancer. A fifth MAb, MN-3,
had properties uniquely different from the NP series of MAb, reacting
strongly with granulocytes but not demonstrating the liquid-phase
ion-sensitivity binding of CEA exhibited by MN-15 and NP-1.

Example 2

Production of Chimeric and Humanized MN-15 Antibodies

[0188] To make a chimeric MN-15 antibody, the murine MN-15 variable region
sequences were attached to human IgG1 constant region sequences, as
described in Leung et al., Hybridoma 13:469 (1994). The sequences of the
murine MN-15 VK (SEQ ID NO:9) and MN-15 VH (SEQ ID NO:10) used
to construct the chimeric MN-15 (cMN-15) are shown in FIG. 2 and FIG. 3.
FIG. 1 shows the results of a competitive binding study of murine vs.
chimeric MN-15, competing with horseradish peroxidase (HRP) labeled
murine MN-15. FIG. 1 demonstrates that the cMN-15 construct has an
affinity for CEA that is virtually identical to the parent murine MN-15
antibody, with a dissociation constant in the nanomolar range. As
expected, the MN-3 (amino terminal) and MN-14 (A3-B3) antibodies, which
bind to different epitopes of CEA than MN-15 (A1B1) did not compete for
binding with HRP-labeled murine MN-15.

[0191] FIG. 4 shows a comparison of the binding affinities of murine,
chimeric and humanized MN-15, in competition with HRP-labeled murine
MN-15. As shown in FIG. 4, the cMN-15 and hMN-15 constructs have
virtually identical binding affinities for CEA with the parental murine
MN-15 antibody, with dissociation constants in the nanomolar range.

[0192] Expression vector DNAs may be transfected into SP2/0 by
electroporation. Transfectomas secreting the various versions of chimeric
or humanized MN-15 are selected and analyzed. For large-scale production,
the cell line is grown in a 16-liter stirred tank bioreactor system
(Sulzer-Chemtech, Woodbury, N.Y.). The culture medium is continuously
harvested from the bioreactor through a 0.2 μm tangential flow
microfiltration hollow fiber unit. The filtered harvest is collected
continuously into a 50-liter reservoir, stored at 4° C., from
which the medium stream is pumped to a protein-A column to collect the
antibody from the product stream. The harvested antibody is purified by a
second pass through a protein-A column and, finally, Q-Sepharose
(Pharmacia, Inc., Piscatway, N.J.). The final product purity is assessed
by SDS-PAGE, immunoelectrophoresis, HPLC, and immunoreactivity.

[0194] CEACAM5 and CEACAM6 are overexpressed in many cancers and are
associated with adhesion and invasion. The effects of three monoclonal
antibodies targeting different epitopes on these antigens
(NH2-terminal [MN-3] and A1B1 domains [MN-15] shared by CEACAM5 and
CEACAM6 and the A3B3 domain [MN-14] restricted to CEACAM5) were evaluated
in migration, invasion, and adhesion assays in vitro using a panel of
human pancreatic, breast, and colonic cancer cell lines, and in the GW-39
human colonic micrometastasis model in vivo. MN-3 Fab' and MN-15 Fab'
were both effective at inhibiting cell migration. MN-15 Fab' treatment
inhibited invasion, reducing cell penetration through an extracellular
matrix (ECM). MN-3 Fab' also decreased invasion but was less effective
than MN-15 Fab' in four of five cell lines. All three monoclonal antibody
Fabs decreased adhesion of tumor cells to endothelial cells by 49% to
58%. MN-15 Fab' but not MN-3 or MN-14 Fabs induced a decrease in adhesion
of three of six cell lines to the ECM protein, fibronectin, but adhesion
to vitronectin, laminin, collagen-I, and collagen-IV was not affected. In
vivo studies showed that treatment with MN-3 Fab' or MN-15 Fab' of mice
implanted with GW-39 human colonic cancer cells increased their survival
(P<0.025 and P<0.01, respectively). These studies show that
antibody Fabs that target either CEACAM5 or CEACAM6 affect cell
migration, cell invasion, and cell adhesion in vitro, and that MN-15 and
MN-3 Fabs have antimetastatic effects in vivo, resulting in improved
survival of mice with metastases. Thus, blocking the N and A1B1 domains
of CEACAM5/CEACAM6 can impede the metastatic process.

[0195] Background

[0196] The eradication of metastatic disease is crucial for achieving
survival in most patients with cancer. The metastatic process consists of
a series of sequential steps, including invasion of extracellular matrix
(ECM), extravasation into vessels, transport in the circulation, adhesion
to endothelial cells in a new tissue, extravasation through the vessel
wall, and migration and proliferation in response to organ-specific
factors at the new site (Fidler, Cancer Res 1990, 50:6130-8). The present
Example deals with the development of an antibody-based therapeutic
approach to impede metastasis.

[0199] Antigenic sites on CEACAM5 and CEACAM6 have been characterized, and
panels of antibodies recognizing specific epitopes have been generated
(Audette et al., Mol Immunol 1987, 24:1177-86; Bjerner et al., Tumour
Biol 2002, 23:249-62). Three subdomains in the N region that are required
for intercellular homotypic adhesion have been identified by
site-directed deletions and point mutations (Taheri et al., J Biol Chem
2000, 275:26935-43). Binding peptides have been developed to these
regions in an effort to block adhesion (Taheri et al., J Biol Chem 2003,
278:14632-9). The concentration of peptide required to block cell
aggregation was 1 mg/mL or a 25-fold higher molar concentration than the
complete antibody, despite the predicted greater penetration of a smaller
peptide. This inefficient dose requirement could be due to instability of
the small peptide or due to its low affinity. We postulated that using
monovalent antibody fragments would be a more effective approach for
targeting these domains, because stability of the MAb will likely be
greater and the affinity higher than corresponding peptides. Our studies
have primarily been done with monovalent fragments of the antibodies of
interest, rather than intact IgGs, because the single binding arm of the
Fab will prevent complexation of tumor cells, which might occur with a
bivalent IgG. In addition, it allowed us to study the efficacy of a
CEA/NCA-90-targeted antibody without the potential effector cell-inducing
activity of the Fc region. We have assessed the expression of CEACAM5 and
CEACAM6 on a panel of tumor cell lines and have determined the effects of
several antibodies targeting different epitopes of CEACAM5 and CEACAM6
for their ability to affect tumor cell migration, invasion, and cell
adhesion in vitro. We have also evaluated the therapeutic potential of
these antibody Fabs in controlling metastasis and survival of mice
bearing a CEACAM5/CEACAM6-expressing human colonic carcinoma.

[0200] Materials and Methods

[0201] Antibody Production

[0202] MN-15 binds to the A1B1 domain (Gold group 4) and MN-3 binds to the
N domain (Gold group 5) found on both CEACAM5 and CEACAM6, while MN-14
binds to the A3B3 domain (Gold group 3) only found on CEACAM5 (FIG. 5).
MN-3 and MN-15 were used as murine MAbs, whereas MN-14 was included in
its humanized form, hMN-14 or labetuzumab (Sharkey et al., Cancer Res
1995, 55:5935-45).

[0206] Cells were harvested from culture and aliquoted into FACSCAN®
tubes containing 2 mL Dulbecco's PBS with 0.2% sodium azide and 1%
appropriate blocking serum. Cells were incubated with 10 mg/mL of hMN-14
anti-CEACAM5 in PBS (+NaN3+1% blocking serum) for 1 hour on ice,
pelleted (1,440 rpm×5 minutes), and supernatant decanted. Some
tubes were then incubated with 10 μg/mL murine MN-15 IgG or MN-3 IgG
(to determine CEACAM6 expression) for 1 hour on ice and pelleted. Neither
MN-15 nor MN-3 bind CEACAM5 once MN-14 is bound so that only CEACAM6 was
detected by MN-15 or MN-3 in the presence of MN-14. Second antibody
conjugated with FITC (FITC-goat-anti-human for CEACAM5 determination and
FITC-goat-anti-mouse for CEACAM6 determination) was added (1:500
secondary antibody in PBS+NaN3+1% blocking serum) for 30 minutes on
ice in the dark. Cells were pelleted, washed twice with 2 mL ice-cold
PBS+NaN3, and resuspended in 1.5% paraformaldehyde. Fluorescence was
read on a flow cytometer. Percentage of cells that were positive and mean
channel fluorescence (MCF) were recorded. CEACAM5 and CEACAM6 expression
on GW-39 tumor xenografts was determined by immunohistochemistry using a
similar approach of hMN-14 followed by biotinylated GAH to detect CEACAM5
and preincubation with hMN-14 followed by mMN-15 or mMN-3 and
biotinylated GAM second antibody to detect CEACAM6 on paraffin sections.

[0207] Spontaneous Migration Assay

[0208] Glass coverslips were placed in 100×15 mm Petri dishes and UV
sterilized overnight. Suspensions of cancer cells (2-4×105
cells/mL) were prepared from 80% to 90% confluent monolayers. Cells were
plated on each coverslip and incubated for 2 to 5 days to reach 70% to
80% confluence. Two diagonal cell-free paths ("wounds") were created by
dragging a sterile yellow pipette tip across the surface. Monolayers were
rinsed several times to remove floating cells and 4 mL of fresh medium
were added back in the absence or presence of antibody IgG or Fab' (10
μg/mL) and incubated at 37° C. After 18 to 24 hours, the medium
was removed and coverslips stained with 1 mL Wright-Giemsa for 1 minute.
The stain was washed off with distilled water, air-dried, and mounted
onto slides with Cytoseal 60. Repopulation of the wound space was
evaluated by counting the number of cells that migrated into the wound
area in 10 representative fields. Regions of migration were photographed
for documentation.

[0209] Endothelial Cell Adhesion Assay

[0210] Human umbilical vein endothelial cells (HUVEC; Cambrex, San Diego,
Calif.) were grown in collagen-coated dishes in EGM Media in a humidified
atmosphere with 5% CO2 at 37° C. At passages 2 to 5, cells
were plated at a density of 4×104 cells per well in 96-well
plates 24 hours before the assay. Interleukin-1β (IL-1β, 1
ng/mL) was added 4 hours before the assay. At the start of the study, the
medium with the IL-1B was removed and fresh DMEM with 1% bovine serum
albumin (BSA) added and incubated for 30 minutes. Fresh medium without
antibodies or with 10 μg/mL of MN-15 Fab', MN-3 Fab', or Ag8 Fab' was
added. Tumor cells (1×106 cells/mL) prelabeled overnight with
3H-thymidine (100 μL per well using 1.0 μCi/mL) were added to
HUVEC cultures and incubated for 30 minutes at 37° C. with
rotation in medium with 20% FBS. Samples were washed thrice with PBS to
remove unattached cells. Attached cells were solubilized with 0.1 N NaOH
and radioactivity was measured in a B scintillation counter. The cpm
attached/total cpm added (attaching potential) was determined.

[0211] Adhesion to Extracellular Matrix Proteins Assay

[0212] The assay was done using the CYTOMATRIX® screening kit from
CHEMICON® (Kit #ECM205, Temecula, Calif.). The kit contains 96-well
plates with strips coated with fibronectin, vitronectin, laminin,
collagen-I, or collagen-IV. Subconfluent cell cultures were used for
these studies. Cells (1×106 cells/mL) were seeded onto coated
substrate and incubated at 37° C. for 1 hour in a CO2
incubator in PBS containing Ca2+/Mg2+ (200 μL per well).
Adherent cells were fixed and stained. The plate was washed to remove
unadhered cells and stained with 100 μL per well of MTS (Cell Titer Aq
96, PROMEGA®, Madison, Wis.) for 5 minutes at room temperature.
Excess stain was removed with three PBS washes. Solubilization buffer
[100 μL of a 50:50 mixture of 0.1 mol/L NaH2PO4 (pH 4.5) and
50% ethanol] was added to each well. Relative attachment was determined
using absorbance readings (A540 nm-A570 nm).

[0213] Collagen-Based Invasion Assay

[0214] The assay was done using CHEMICON® Kit (#ECM551,
MILLIPORE®, Billerica, Mass.). Tumor cells at 80% confluence and were
serum-starved for 18 to 24 hours before the assay was used. Cells were
harvested with 5 mL of 2 mmol/L EDTA/PBS per 100-mm dish and incubated at
37° C. for 5 to 15 minutes. Cells were collected into 10 to 20 mL
of quenching medium (serum-free DMEM containing 5% BSA) to inactivate
trypsin/EDTA from the harvesting buffer. Cells were pelleted, resuspended
in quenching medium (1×106 cells/mL), and appropriate antibody
Fabs were added to individual cell aliquots. Cell invasion potential was
determined as follows. Baseline invasion and invasion in the presence of
a chemoattractant (10% FBS) were measured after a 72-hour incubation
period at 37° C. in a 5% CO2 incubator. The bottom side of
the collagen-coated polycarbonate membrane insert of the invasion chamber
was placed in 400 μL of cell stain for 20 minutes at room temperature
and washed. The dye was extracted and transferred into a 96-well
microtiter plate for colorimetric measurements (A560 nm).

[0215] In Vivo Therapy Studies

[0216] Female athymic nude mice (6 to 8 weeks old) were purchased from
TACONIC® (Germantown, N.Y.). Therapy studies were done using our
CEACAM5+/CEACAM6+GW-39 intrapulmonary micrometastasis model, GW-39iv
(Sharkey et al., J Natl Cancer Inst 1991, 83:627-32; Blumenthal et al.,
Cancer Res 1992, 52:6036-44). The GW-39 human colonic carcinoma has been
maintained as a serially transplanted signet-ring cell cancer line since
1966 (Goldenberg et al., Transplantation 1966, 4:760-4). Stock s.c. GW-39
tumors grown in nude mice were used to prepare a 10% cell suspension.
Cells (30 μL) were injected i.v. into the caudal vein of 5- to
6-week-old female nude mice. This results in ˜50 to 100 tumor
nodules developing in the lungs and a median survival time of 7 to 9
weeks. Cells were pretreated with antibody in vitro (10 μg/mL) before
implantation, and then mice were given one additional dose (100 μg per
mouse i.v.) 1 day after implantation. Body weight was monitored weekly
and animal survival recorded. Results were analyzed by the Kaplan-Meier
estimated survival curves, and significance was determined with the
log-rank comparison of survival curves. Median survival time for each
treatment group also was determined. All studies used 10 mice per
treatment group.

[0217] Results

[0218] CEACAM5 and CEACAM6 Expression in Cell Lines

[0219] Flow cytometric analysis of CEACAM5 and CEACAM6 expression in a
panel of 31 to 33 commonly used solid tumor cell lines revealed that only
6 of 29 (20.7%) expressed significant amounts of CEACAM5, whereas 16 of
30 (53.3%) lines were positive for CEACAM6 (Table 1). Two cancer cell
lines were CEACAM5+/CEACAM6- (Moser and LNCAP), four were
CEACAM5+/CEACAM6+, and 12 were CEACAM5-/CEANCAM6+. The CEACAM6+ cell
lines included 7 of 10 breast cancers, one of four ovarian cancers, three
of four colon cancers, three of four pancreatic cancers, one of six
prostate cancers, and two of four non-small cell lung cancer lines. Many
of these tumor lines had >95% of cells expressing the CEACAM6 antigen
and, in some cases, expression was very high (MCF of 641 for ZR75-30, 702
for BXPC3, and 476 for CaPAN-1). In contrast, the most positive
CEACAM5-expressing lines had <60% of cells expressing the antigen,
with a MCF<120.

[0220] Effect of Anti-CEACAM5 and Anti-CEACAM6 Antibodies on Cell
Migration

[0221] Using the wound-healing assay, we assessed cell migratory activity
in vitro in a panel of cell lines. LS174T and HT-29 cells showed the most
migratory activity, whereas ZR75-30, MCF-7, and BXPC3 cells showed very
little migration (results not shown). Compared with an irrelevant
antibody, MN-3 and MN-15 intact IgG and Fab' all reduced migration in
both cell lines. In HT-29, the number of migrating cells per field
decreased from 14.9±9.1 to 6.1±0.9 and 3.7±1.6 cells with MN-3
IgG and Fab', respectively, and to 5.5±1.8 and 5.4±0.8 with MN-15
IgG and Fab', respectively (P<0.01 for all comparisons of MAb
treatment with untreated cells; n=10 for each arm). In LS174T studies,
untreated cells had 38.8±10.9 migrating cells per field, whereas cells
treated with MN-3 IgG or Fab' had 21.4±5.2 and 18.1±1.6 migrating
cells, respectively (P<0.001 versus untreated), and cells treated with
MN-15 IgG or Fab' had 20.3±0.1 and 21.6±1.2 migrating cells,
respectively (P<0.001 versus untreated). Thus, blocking either the N
or A1B1 domain of CEACAM6 with either an intact IgG or a monovalent Fab
inhibited migration in vitro.

[0222] Effect of MN-15 and MN-3 on Tumor Cell Adhesion to Endothelial
Cells

[0223] Because both CEACAM5 and CEACAM6 are known to have an adhesion
role, we evaluated whether blocking these antigens reduces adhesion to
endothelial cells (FIG. 6). In the CEACAM5-/CEACAM6- MCA38 murine colonic
cancer line, neither MN-3 Fab' targeting the N domain nor MN-15 Fab'
targeting the A1B1 domain of CEACAM6 had an effect on tumor
cell-endothelial cell adhesion (not shown). In contrast, both antibodies
reduced endothelial cell binding of MCA38cea (a human CEA-transfected
line) from 11.68±0.77% to 6.42±2.1% (MN-3) and 5.53±1.15%
(MN-15), being significant (P<0.05) for both Fabs (FIG. 6). Both
antibodies induced a 49% to 58% adhesion-inhibition in four other cell
lines (P<0.01 for MN-3 Fab' on MCF-7, HT-29, and BT-20; P<0.02 for
MN-15 on the same three lines; and P<0.05 for both Fabs on Moser cell
adhesion to endothelial cells, FIG. 6). An isotype-matched irrelevant
antibody Fab' (Ag8) did not affect tumor cell-endothelial cell adhesion
nor did the MN-14 anti-CEA Fab'. The magnitude of the anti-adhesion
effect did not seem strictly correlated with the amount of CEACAM5 or
CEACAM6 expressed, because MN-15 Fab' resulted in a greater decrease in
adhesion in HT-29 cells (48%) than with MCF-7 cells (41%) that express
much more CEACAM6.

[0225] In addition to tumor cell binding to endothelial cells, these cells
can also bind ECM proteins. The extent of tumor cell binding to ECM
proteins varies among different cell lines. MCF-7 bound well to four of
five proteins (A560>1.1) except laminin (A560
nm=0.2±0.04), whereas ZR75-30 attached weakly to all five ECM proteins
evaluated (A560 nm<0.45). MDA-468 bound quite well to collagen-I
and collagen-IV (A560 nm=1.25±0.07 and 0.97±0.03,
respectively) but not as well to fibronectin, vitronectin, or laminin.
The reverse pattern was seen with CaPAN-1 (A560 nm=1.78±0.21 for
fibronectin, 0.88±0.11 for vitronectin, 1.14±0.09 for laminin,
0.07±0.00 for collagen-I, and 0.13±0.08 for collagen-IV). None of
the antibodies (IgG or Fab') modulated adhesion to vitronectin, laminin,
collagen-I, or collagen-IV (results not shown). However, MN-15 IgG
decreased adhesion of MCF-7, MDA-468, and CaPAN-1 to fibronectin by 29%
(P<0.01), 51% (P<0.001), and 47% (P<0.02), respectively, while
not affecting the binding of ZR75-30, BXPC3, or Moser to fibronectin. Two
of these three MAb-unresponsive cell lines had the lowest baseline
adhesion to fibronectin.

[0226] Effect of MN-3, MN-15, and MN-14 Fabs on Tumor Cell Invasion

[0227] Specific invasion in response to FBS as a chemoattractant was
1.9-fold (LS174T) to 7.4-fold (BXPC3) higher than in the absence of FBS
(not shown). MN-15 Fab' was more effective than MN-3 Fab' at reducing
tumor cell invasion in vitro in five cell lines that expressed CEACAM5,
CEACAM6, or both antigens. MN-14 anti-CEA (CEACAM5) had no effect on
tumor cell invasion in most cell lines. MDA-231 expresses neither
antigen, and its invasion was not reduced by either MN-3 or MN-15 IgG or
Fab'. For the five antigen-positive lines, both the intact IgG and the
monovalent Fab' for a given antibody were equally effective. For example,
MN-15 Fab' reduced cell invasion of LS174T, MCF-7, GR75-30, BXPC3, and
CaPAN-1 cells by 30% (P<0.02), 77% (P<0.01), 49% (P<0.01), 44%
(P<0.01), and 73% (P<0.002), respectively. The effect of MN-3 Fab'
on the same five lines was a reduction in invasion of 3% (P═NS), 47%
(P<0.01), 59% (P<0.01), 0% (P═NS), and 55% (P<0.05),
respectively. Thus, the A1B1 domain of CEACAM6 seems to be a more
important target than the N domain for the process of tumor cell
invasion.

[0229] GW-39 expresses both CEACAM5 and CEACAM6, as shown by
immunohistochemistry (FIG. 7, top). Based on the in vitro results
suggesting that anti-CEACAM6 antibodies had limited antiproliferative
effects yet showed significant anti-invasive and anti-adhesive
properties, we pretreated GW-39 tumor cell suspensions with each of the
antibody fragments (10 μg/mL) for 15 minutes before i.v. injection of
304 of cells and then dosed with 100 μg of antibody on the day after
transplantation. This design simulates the effect of a continuous
exposure to antibody that would be available at any time that a cell from
a primary tumor might initiate the metastatic cascade. The results
presented in FIG. 7 illustrate that both MN-15 and MN-3 Fabs increased
median survival time (>77 and 77 days, respectively; P<0.001 versus
untreated cells) of these mice, whereas hMN-14 Fab' (49 days) did not
affect survival significantly (42 days for untreated mice). Although the
study was continued until mice were near death, rather than sacrificing
them at a defined time after treatment and counting the number of lung
nodules, median survival time should correlate with amount of lung
metastatic disease. The results with hMN-14 Fab' in this study are
similar to what we reported for hMN-14 F(ab)2 in this metastatic
model (Blumenthal et al., Cancer Immuno Immunother 2005, 54:315-27),
indicating that targeting the A3B3 domain does not affect median survival
in this model, whereas targeting the N and A1B1 domains shared by CEACAM5
and CEACAM6 did affect metastasis and host survival when the respective
Fabs are given.

[0230] Discussion

[0231] A long-term goal of immunotherapy has been to induce antitumor
responses against tumor-related antigens. Antibodies that directly
perturb signaling mechanisms have shown clinical benefit, such as those
directed against the extracellular domains of HER-2/neu, epidermal growth
factor receptor, and CD20 (Weiner & Adams, Oncogene 2000, 19:6144-51;
Alas et al., Cancer Res 2001, 61:5137-44). The studies presented herein
suggest that instead of being directly therapeutic by immune effector or
direct cytostatic mechanisms, MAbs against CEACAM6 inhibit migration,
invasion, and adhesion, thereby limiting metastasis. The monovalent Fab'
form was used for most of our studies to avoid effector cell function
from the Fc region of the MAb and focus on mechanisms implicated in the
metastatic process.

[0232] CEACAM5 is overexpressed in a majority of carcinomas, including
those of the gastrointestinal, respiratory and genitourinary systems, and
the breast. Our results show that another CEA family member, CEACAM6, may
be as good, if not better, as a target for solid tumor antimetastatic
therapy. We have shown that CEACAM6 is expressed in a larger percentage
of solid tumor cell lines than CEACAM5, and the high MCF on many of these
lines suggests that more anti-CEACAM6 MAb can be delivered to these tumor
cells. Thus, the MN-15 and MN-3 MAbs are useful for tumors that express
CEACAM6 or CEACAM5, because they target epitopes that are shared by both
antigens. These MAbs may therefore have advantages over MAbs like MN-14,
which only target CEACAM5, or MAbs that are specific for only CEACAM6 and
do not cross-react with CEACAM5. Our data are consistent with previous
reports that showed a relatively high level of CEACAM6 in the sera of a
large number of lung, liver, pancreatic, breast, and colorectal cancer
patients (Kuroki et al., Anticancer Res 1999, 19:5599-606).
Interestingly, some patients were CEACAM5-/CEACAM6+, further suggesting
that CEACAM6 might be a useful independent target.

[0233] It is known that CEACAM6 is expressed on the surface of
neutrophils, thus modulating adherence to endothelial-leukocyte adhesion
molecule-1 on activated endothelial cells (Kuijpers et al., J Cell Biol
1992, 118:457-66). We have shown that MAbs to different epitopes on
CEACAM6 (N and A1B1 domains) affect cell adhesion with endothelial cells.
There is also evidence that CEACAM5 can affect cell adhesion to
endothelial cells (Gangopadhyay et al. Clin Exp Metastasis 1998,
16:703-12) via activation of Kupffer cells and stimulation of IL-1β,
tumor necrosis factor-α, and IL-6 production. These cytokines then
induce the expression of intercellular adhesion molecules on endothelial
cells, thus increasing adhesion of tumor cells to endothelium. Our
results have shown that MN-3 and MN-15 Fab' s are more active than MN-14
Fab' at reducing adhesion of CEACAM5+/CEACAM6+ tumor cells to endothelial
cells. Thus, both the N and the A1B1 domains but not the A3B3 domain are
involved in tumor cell to endothelial cell adhesion.

[0234] Tumor cells can adhere to other tumor cells, to endothelial cells,
as well as to ECM proteins. We have found that the amount of adhesion to
a panel of these proteins varied between cell lines and was not related
to the type of tumor (e.g., breast and pancreatic) or to the amount of
CEACAM5 or CEACAM6 expressed. Tumor cell interactions with ECM proteins
are important for migration and invasion and therefore metastasis. For
example, tumor cell interactions with fibronectin are involved in the
development of secondary tumors inside the bone marrow stroma via the
α5β1 integrin (Van der Velde-Zimmermann et al., Exp Cell Res
1997, 230:111-20). Blocking adhesion with polypeptide fragments of
heparin-binding domains of fibronectin inhibited metastasis (Matsumoto et
al., Jpn J Cancer Res 1991, 82:1130-8). Similar results have been
obtained with a peptide blocking tumor cell-laminin adhesion (Islam et
al., Surgery 1993, 113:676-82). Surprisingly, only MN-15 (A1B1 domain)
was able to reduce adhesion to fibronectin in three of six tumor cell
lines tested. The percent inhibition in fibronectin adhesion in this
panel of cell lines did not correlate with the amount of adhesion in
untreated cell samples. MAbs targeting the N or A3B3 domain did not
affect cell adhesion to fibronectin. In one report by Duxbury et al. (J
Biol Chem 2004, 279:23176-82), MAb-mediated CEACAM6 cross-linking
resulted in increased ECM adhesion. However, the targeted epitope was
different from the ones studied here.

[0235] Active migration of tumor cells is a prerequisite of tumor cell
invasion and metastasis. Adhesion molecules that increase invasion also
enhance the migratory process (Hazan et al., J Cell Biol 2000,
148:779-90). Overexpression of CEACAM6 has been reported to promote
cellular invasiveness of pancreatic cancer (Duxbury et al., Oncogene
2004, 23:465-73). Agents that inhibit metastases often affect several
steps including migration, adhesion, and invasion. Because our data
suggest that CEACAM6 has a role in adhesion and invasion, it is important
to also assess the ability of these MAbs to impede migration. We have
shown that in cell lines with strong migratory tendencies, MAb blocking
of CEACAM5 and/or CEACAM6, with MN-3>MN-15, decreases the number of
migrating cells. In our in vitro assay, the process of cell invasion,
which involves adhesion to ECM and migration steps, was inhibited by
MN-15 Fab'>MN-3 Fab', suggesting that the A1B1 domain of CEACAM6 is
more important for this step but that the N domain also plays a role.

[0236] One of the notable advantages of MN-15 or MN-3 MAb therapy,
compared with our previously reported results with MN-14 anti-CEA IgG
(Blumenthal et al., Cancer Immunol Immunother 2005, 54:315-27), is the
ability to target tumors that express either CEACAM6, CEACAM5, or both,
whereas MN-14 can only be used for CEACAM5+ tumors. As shown in Table 1,
many solid tumor lines express CEACAM6 but not CEACAM5 or express more
CEACAM6 than CEACAM5. These tumor types are candidates for
metastasis-directed MAb therapy with CEACAM6 MAbs.

[0237] An important consideration based on the in vivo experiments is the
availability of MAb when cells first enter the circulation. MN-15 Fab'
and MN-3 Fab' showed therapeutic efficacy if cells were exposed to MAb
before the initiation of the metastatic process. However, if MAb was
delivered after cancer cells had exited the vasculature and had begun to
seed in the lung, MAbs alone were not therapeutic (data not shown).
Therefore, in certain preferred embodiments, anti-CEACAM5/CEACAM6 MAbs
would be available continuously, perhaps using implantable pumps, to
maintain a desired level in the circulation.

[0238] Overall, the anti-metastasis and MAb inhibition of adhesion,
invasion, and migration is a technology that should be relatively
nontoxic, not limited by issues of drug resistance, and easy to apply as
an adjuvant with other standard and/or experimental therapy approaches.
Because CEACAM6 is also expressed in normal lung, spleen, and
granulocytes (Grunert et al., Int J Cancer 1995, 63:349-55), the effect
of anti-CEACAM6 MAb on normal tissues is one consideration for
therapeutic use. In one report, CEACAM6-targeted immunotoxin therapy was
effective in a tumor-bearing nude mouse model (Duxbury et al., Biochem
Biophys Res Commun 2004, 317:837-43), but this model does not express
CEACAM6 on normal tissues.

[0239] In summary, we have shown that anti-CEACAM6/CEACAM5 MAb fragments
devoid of effector cell functions and targeting the N and A1B1 domains of
these antigens block migration, adhesion to endothelial cells and ECM,
and invasion, and also increase the median survival of mice with
intrapulmonary micrometastases of human colonic cancer. These results
indicate that antibodies against CEACAM5 and CEACAM6, such as MN-15, may
be efficacious in human cancer therapy.

Example 4

Expression Patterns of CEACAM5 and CEACAM6 in Primary and Metastatic
Cancers

[0240] Summary

[0241] Many breast, pancreatic, colonic and non-small-cell lung carcinoma
lines express CEACAM6 (NCA-90) and CEACAM5 (carcinoembryonic antigen,
CEA), and antibodies to both can affect tumor cell growth in vitro and in
vivo. Here, we compare both antigens as a function of histological
phenotype in breast, pancreatic, lung, ovarian, and prostatic cancers,
including patient-matched normal, primary tumor, and metastatic breast
and colonic cancer specimens.

[0242] Antigen expression was determined by immunohistochemistry (1HC)
using tissue microarrays with MN-15 and MN-3 antibodies targeting the
A1B1- and N-domains of CEACAM6, respectively, and the MN-14 antibody
targeting the A3B3 domain of CEACAM5. IHC was performed using
avidin-biotin-diaminobenzide staining. The average score±SD
(0=negative/8=highest) for each histotype was recorded.

[0243] For all tumors, the amount of CEACAM6 expressed was greater than
that of CEACAM5, and reflected tumor histotype. In breast tumors, CEACAM6
was highest in papillary>infiltrating ductal>lobular>phyllodes;
in pancreatic tumors,
moderately-differentiated>well-differentiated>poorly-differentiated
tumors; mucinous ovarian adenocarcinomas had almost 3-fold more CEACAM6
than serous ovarian adenocarcinomas; lung adenocarcinomas>squamous
tumors; and liver metastases of colonic carcinoma>primary tumors=lymph
nodes metastases>normal intestine. However, CEACAM6 expression was
similar in prostate cancer and normal tissues. The amount of CEACAM6 in
metastatic colon tumors found in liver was higher than in many primary
colon tumors. In contrast, CEACAM6 immunostaining of lymph node
metastases from breast, colon, or lung tumors was similar to the primary
tumor.

[0244] CEACAM6 expression is elevated in many solid tumors, but variable
as a function of histotype. Based on previous work demonstrating a role
for CEACAM6 in tumor cell migration, invasion and adhesion, and formation
of distant metastases (Blumenthal et al., Cancer Res 65: 8809-8817,
2005), it may be an important target for antibody-based therapy.

[0245] Background

[0246] The human carcinoembryonic antigen (CEA) family has 7 genes
belonging to the CEACAM subgroup. These subgroup members are mainly
associated with the cell membrane and show a complex expression pattern
in normal and cancerous tissues. The CEACAM5 gene, also known as CD66e,
codes for the protein, CEA (Beauchemin et al., Exp Cell Res. 1999,
252:243-249). CEACAM5 was first described in 1965 as a gastrointestinal
oncofetal antigen (Gold & Freedman, J Exp Med. 1965, 122:467-481), but is
now known to be overexpressed in a majority of carcinomas, including
those of the gastrointestinal tract, the respiratory and genitourinary
systems, and breast cancer (Goldenberg et al., J Natl Cancer Inst. 1976,
57:11-22; Shively et al., Crit. Rev Oncol Hematol. 1985, 2:355-399;
Hammarstrom, Semin Cancer Biol. 1999, 9:67-81). CEACAM6 (also called
CD66c or NCA-90) is a non-specific cross-reacting glycoprotein antigen
that shares some antigenic determinants with CEACAM5 (Kuroki et al.,
Biochem Biophys Res Comm. 1992, 182:501-506). CEACAM6 also is expressed
on granulocytes and epithelia from various organs, and has a broader
expression zone in proliferating cells of hyperplastic colonic polyps and
adenomas, compared with normal mucosa (Scholzel et al., Am J Pathol.
2000, 157:1051-1052), as well as by many human cancers (Kuroki et al.,
Anticancer Res. 1999, 19:5599-5606; Hinoda et al. J Gastroenterol. 1997,
32:200-205). Relatively high serum levels of CEACAM6 are found in
patients with lung, pancreatic, breast, colorectal, and hepatocellular
carcinomas. The amount of CEACAM6 does not correlate with the amount of
CEACAM5 expressed (Kuroki et al., Anticancer Res. 1999, 19:5599-5606).

[0247] Expression of CEACAM6 in colorectal cancer correlates inversely
with cellular differentiation (Ilantzis et al., Neoplasia. 2002,
4:151-163) and is an independent prognostic factor associated with a
higher risk of relapse (Jantscheff et al., J Clin Oncol. 2003,
21:3638-3646). Both CEACAM5 and CEACAM6 have a role in cell adhesion,
invasion and metastasis, as discussed in Example 3. The present study
used tissue microarray analysis to compare the relative expression of
CEACAM5 and CEACAM6 in different histotypes of solid tumors, and compared
expression between primary sites and matched metastases in the same
patients.

[0248] Methods

[0249] Antibodies

[0250] As discussed above, MN-15 binds to the A1B1-domain and MN-3 binds
to the N-domain found on both CEACAM5 and CEACAM6. MN-14 binds to the
A3B3 domain only found on CEACAM5. These antibodies have similar
affinities for their target antigens (Hansen et al., Cancer. 1993,
71:3478-3485). MN-3 and MN-15 were used as murine MAbs, while MN-14 was
included in its humanized form, hMN-14 (labetuzumab).

[0254] Slides were deparaffinized in xylene, rehydrated, and treated with
fresh 0.3% hydrogen peroxide in methanol for 15 min. Following a wash in
1× phosphate-buffered saline (PBS, pH 7.4), slides were blocked
with normal serum in a humid chamber for 20 min at room temperature (RT).
Excess serum was rinsed off with 1×PBS and slides were incubated in
a humid chamber with 25-50 μl of primary antibody (10 μg/ml) for 45
min at RT. For CEACAM5 staining, the primary antibody was murine mMN-14
IgG. For CEACAM6 staining, slides were first blocked with humanized
hMN-14 IgG and then incubated with primary antibody, either murine mMN-15
or mMN-3 IgG. Excess primary antibody was washed off and sections were
covered with biotinylated goat-anti-mouse for 30 min in a humid chamber
at RT. Slides were then flooded with 0.3% H2O2 in methanol and
25 μl avidin-horseradish peroxidase (HRP) conjugate was added. Slides
were incubated for 45 min at RT, washed in 1×PBS, and covered with
100 μl 3,3'-diaminobenzidine tetrahydrochloride solution (100 mg/ml
diaminobenzide in 0.1 M sodium acetate buffer, pH 6.0, with 0.01% (v/v)
H2O2) for 15 min. Slides were washed twice by dipping in tap
water and counterstained with 4 quick dips in hematoxylin (filtered
through WHATMAN® #4 filter paper). Slides were rinsed, air-dried, and
mounted with 1-2 drops of cytoseal and a glass coverslip. The method of
Kawai was used to calculate a semi-quantitative score from 0 to 8 for
staining of each tissue core (Kawai et al., Clin Cancer Res. 2005,
11:5084-5089). The number of positive cells/file was estimated and
assigned a number: 0=none, 1= 1/100 cells, 2= 1/100 to 1/10 cells, 3=
1/10 to 1/3 cells, 4=1/3 to 2/3 cells, and 5=>2/3 cells. The intensity
of staining was then determined where 0=none, 1=weak, 2=intermediate, and
3=strong. The first and second scores were then added together resulting
in a maximum staining score of 8 for any tissue core. Two independent
blinded investigators performed IHC analysis and results were strongly
consistent between the two readings. Results were recorded as the
mean±standard deviation for each group. Comparisons between CEACM5 and
CEACAM6 scores for a given histotype or between histotypes for each
antigen were assessed by a one-factor analysis of variance with the use
of a two-tailed F test and a 95% confidence limit. The null hypothesis
Ho: μ1=μ2=1/4 μk, where k equals the number of experimental
groups, was used. A two-tailed test takes into account an extreme value
in any one group that deviates from the population mean in either the
high or low direction (two-sided). The F value is a measure of the
probability that this difference in groups could occur by chance alone.

[0255] Results

[0256] Expression in Solid Tumors as a Function of Histotype

[0257] For all tumor cores evaluated, the amount of CEACAM6 was greater
than that of CEACAM5. However, the homogeneity of expression and staining
intensity varied between tissue histotypes and between samples within the
same histological type. We evaluated 45 breast tumor cores: 30
infiltrating ductal carcinoma, 8 papillary, 4 lobular, and 3 phyllodes.
CEACAM6 levels were higher than CEACAM5 levels for all histotypes
(P<0.001). The highest CEACAM6 expression was found in papillary
(6.0±2.1)>infiltrating ductal (5.1±2.5)>lobular
(4.0±0.8)>phyllodes (2.0±1.0). The differences between papillary
and lobular breast cancers were significant at the P<0.01 level. The
highest CEACAM5 expression was found in papillary samples (1.4±1.4),
but was not statistically different from infiltrating ductal or lobular
samples. Pyllodes breast cancer is a stromal tumor, usually benign, and
should therefore not express CEACAM5 or CEACAM6.

[0258] CEACAM5 and CEACAM6 expression was assessed in 6 different lung
cancers: 5 each of well, moderately and poorly differentiated
adenocarcinoma, 5 each of well, moderately and poorly differentiated
squamous carcinoma, 3 each of large cell and bronchioalveolar, and 2 each
of large cell neuroendocrine and small cell cancer. Among these,
adenocarcinoma expressed more CEACAM6 than squamous cancer (P<0.001).
The highest CEACAM6 expression was found in moderately-differentiated
adenocarcinoma (7.8±0.4)>well-differentiated adenocarcinoma
(7.3±1.1)=bronchioalveolar (7.2±0.8)>poorly-differentiated
adenocarcinoma (6.8±1.0)>small-cell
(5.5±0.7)>well-differentiated squamous
(5.2±1.0)>moderately-differentiated squamous cancer (4.9±1.1).
CEACAM6 levels in large-cell (4.5±0.9) and poorly-differentiated
squamous carcinomas (3.8±1.3) were similar to non-neoplastic lung
tissue (P═NS), suggesting that anti-CEACAM6 antibodies would not be
effective with these histotypes of lung cancer. The highest expression of
CEACAM5 was in small-cell lung cancer specimens (5.5±0.7), followed by
large-cell neuroendocrine tumors (4.75±3.18). Large-cell tumors were
CEA-negative and all adenocarcinomas and serous tumors scored
≦2.60.

[0259] Pancreatic cancer has been the most extensively studied neoplasm
with respect to CEACAM6 expression. We evaluated CEACAM5 and CEACAM6 in
pancreatic cancer as a function of tumor cell differentiation. One
well-differentiated, 3 well-moderately differentiated, 13
moderately-differentiated, 2 moderately- to poorly-differentiated, and 7
poorly-differentiated tumor cores were studied. The highest expression of
CEACAM6 in pancreatic tumors was found in
moderately-(7.5±0.7)>moderately-poor (5.9±1.9)=well-moderately
differentiated (5.8±1.8)>poorly-differentiated tumors
(5.1±2.5)>well-differentiated (4.0±0.0) adenocarcinomas
(P═NS between the subtypes). Non-neoplastic pancreas CEACAM6
expression was 2.25±0.5. The well-moderately, moderately, and
moderately-poor adenocarcinomas were significantly higher than
non-neoplastic pancreas (P<0.001). CEACAM6 expression did not
correlate with disease stage. Samples with high (8) and low (3-4)
expression could be found in stages IA-IB, IIA-IIB, and IV. CEACAM5
expression was lower than CEACAM6 for all histotypes; the highest
expression being found in moderately differentiated tumors (4.0±1.4)
and the least in the moderate-poor (0.92±1.92) and
poorly-differentiated (1.4±1.5) tumors. Only the moderately and the
well-moderately differentiated tumors expressed significantly more
CEACAM5 than non-neoplastic tissues (P<0.002 and P<0.005,
respectively).

[0260] Eighteen stage-II, 15 stage-III, and 4 stage-IV prostate tumor
cores were stained for CEACAM5 and CEACAM6. Gleason scores of 4 to 9 were
represented in the stage-II samples, and Gleason scores of 6 to 10 were
found in the stage-III specimens. All stage-IV samples were Gleason 9-10.
Expression did not correlate with Gleason score of the sample within any
stage. Similar expression of CEACAM6 was found in stage-II, -III, and -IV
prostate cancer (3.3-3.8), and was not significantly different from
non-neoplastic prostate tissue (P═NS). CEACAM5 expression was
consistently below 0.9 for all stages of prostate cancer and was not
greater than expression levels in non-neoplastic prostate tissue
(0.5±1.0; P═NS).

[0263] CEACAM6 expression has been associated with cell adhesion, a key
step in the metastatic cascade. We have shown above that antibody to
CEACAM6 expression can block adhesion. Therefore, we assessed whether
CEACAM6 expression was similar or different between matched primary colon
and metastatic liver sites. In half of the matched cases (N=6), CEACAM6
expression was much greater in the liver metastasis than in the primary
colon tumors, and in the remaining 6 cases, the amounts were comparable
between the primary and the metastatic liver sites.

[0264] In contrast to the higher expression of CEACAM6 in many secondary
liver sites from colon cancer, there was no pattern for CEACAM6
expression between primary tumor and lymph node metastases. For breast
samples, the lymph node sites had higher CEACAM6 expression in 7 pairs,
lower CEACAM6 in 6, and no difference in 25 pairs. For lung samples, the
lymph node sites had higher CEACAM6 expression in 10 pairs, lower CEACAM6
in 11, and no difference in 16 pairs. For colon samples, the lymph node
sites had higher CEACAM6 expression in 7 pairs, lower CEACAM6 in 10, and
no difference in 11 pairs.

[0267] Studies have shown that CEACAM5 affects expression of various
groups of cancer-related genes, especially cell cycle and apoptotic
genes, protecting colonic tumor cells from various apoptotic stimuli,
such as treatment with 5-fluorouracil (Soeth et al., Clin Cancer Res.
2001, 7:2022-2030). Therefore, CEACAM5 expression may be a means for
cancer cells to overcome apoptosis-inducing therapies. Ordonez et al.
have reported that expression of both CEACAM5 and CEACAM6 plays a role in
inhibiting apoptosis of cells when deprived of their anchorage to the
extracellular matrix, a process known as anoikis (Ordonez et al., Cancer
Res. 2000, 60:3419-3424). Increased expression of CEACAM6 correlates with
a decrease in sensitivity to drugs, like gemcitabine (Duxbury et al.,
Cancer Res. 2004, 64:3987-3993). Targeting CEACAM5 and/or CEACAM6 may
therefore be a novel method of modulating cancer cell chemosensitivity
and apoptosis. It has been reported that siRNA to CEACAM6 impairs
resistance to anoikis and increases caspase-mediated apoptosis of
xenografted tumors (Duxbury et al., Oncogene. 2004, 23:465-473).
Antibody-directed targeting of CEACAM6 may provide a clinically feasible
alternative to RNA interference silencing to enhance responsiveness to
chemotherapeutic agents in those tumors that express CEACAM6.

[0268] To determine which solid tumors and histological types would be
most amenable to antibody blocking of CEACAM5 and CEACAM6, we studied
expression of these antigens using tissue microarray analysis. To date,
pancreatic and colonic cancer have been the focus of CEACAM6 expression
in the literature (Duxbury et al., Ann Surg. 2005, 241:491-496; Kodera et
al., Br J Cancer. 1993, 68:130-136). Here, we have further explored the
expression of CEACAM6 in a panel of solid tumors: breast, lung, ovary and
prostate cancer, in addition to expanding on pancreatic and colonic
tumors, and used tissue microarrays to further define tumors that are
CEACAM6+ as a function of histological type in all six solid tumor
categories. Our results show that expression is strongly dependent on the
histotype of the tumor. Antigen expression in some subtypes is 2-4-fold
higher than in normal tissues, while in others, expression is similar to
non-neoplastic tissues.

[0269] The demonstration of higher CEACAM6 expression compared with
CEACAM5 across most solid tumors, and the differential expression as a
function of histotype, are important observations for translating
anti-CEACAM6 therapy to patients. This analysis is a step towards
elucidating the importance of CEACAM6 as a tumor target in a variety of
solid tumors that extend the many important studies reported for
pancreatic cancer (Duxbury et al., J Biol. Chem. 2004, 279:23176-23182;
Duxbury et al., Cancer Res. 2004, 64:3987-3993; Duxbury et al., Biochem
Biophys Res Comm. 2004, 317:837-843; Duxbury et al., Ann Surg. 2005,
241:491-496). It also reveals that expression level varies as a function
of tumor histotype.

[0270] We have also addressed the expression pattern of CEACAM6 in primary
tumors and in matched metastases in the same patients. Our results show
that in half of the clinical specimens, liver metastases had a much
higher expression of CEACAM6 than the primary colorectal tumors,
suggesting that in such patients, blocking adhesion and invasion that
results from CEACAM6 expression might influence the ability of tumor
cells to metastasize, as we have in fact shown experimentally (Goldenberg
et al., J Natl Cancer Inst. 1976, 57:11-22). However, CEACAM6 expression
in lymph node metastases was similar to the amount of antigen in primary
breast, colon or lung tumor samples. The mechanism by which malignant
tumors invade lymphatics and metastasize to regional lymph nodes appears
to be regulated by VEGF-C and VEGF-D induced lymphogenesis (Detmar &
Hirakawa, J Exp Med. 2002, 196:713-718) and a chemokine gradient.
Directional movement is related to chemokine receptor expression on tumor
cells (Nathanson, Cancer. 2003, 98:413-423), but does not involve members
of the CEACAM family. In contrast, CEACAM6 plays an important role in
migration, invasion and adhesion (Duxbury et al., Oncogene. 2004,
23:465-473), steps that are important in the metastatic spread to
secondary tissue sites other than lymph nodes. Anti-adhesive molecules
that disrupt cell-matrix and cell-cell attachments have been proposed as
potential cancer therapeutics based on their ability to interfere with
motility, adhesion, and metastatic progression (Blumenthal et al., Cancer
Res. 2005, 65:8809-8817; Glinsky, Cancer Metastasis Rev. 1998,
17:177-186; Kerbel et al., Bulletin de I'institut Pasteur. 1995,
92:248-256).

[0271] We have reported that the humanized anti-CEA (CEACAM5) antibody,
hMN-14, can enhance the therapeutic effects of two cytotoxic drugs used
frequently in colorectal cancer therapy, fluorouracil and CPT-11, in both
subcutaneous and metastatic human colonic tumor cells propagated in nude
mice (Blumenthal et al., Cancer Immuno Immunother. 2004, 54:315-27). In
another high CEA-expressing human medullary thyroid cancer xenograft, we
have also shown that MN-14 anti-CEA IgG can inhibit tumor cell growth and
also augment the effects of dacarbazine, a drug that is active in this
cancer type (Stein et al., Mol Cancer Ther. 2004, 2:1559-1564). One
explanation may involve a role in antibody blocking adhesion (Zhou et
al., Cancer Res. 1993, 53:3817-3822) and thereby chemosensitizing the
tumor cells.

[0274] Based on expression level, CEACAM6 may be a more promising target
for antibody-based anti-metastatic and chemosensitizing therapy than
CEACAM5 in the solid tumors studied. Furthermore, CEACAM6 may be a useful
antigen to target in select subtypes of solid tumors. In colonic cancer,
CEACAM6 may play an important role in the development of distant
metastases.

Example 5

In Vivo Effect of Pretreatment with an Immunomodulator Prior to Treatment
With hMN-15 and CPT-11 on Tumor Cell Chemosensitivity

[0275] The effect of combined treatment of hMN-15 with CPT-11 is
evaluated, initiated together in mice with GW-39 tumors expressing higher
CEA levels, as a result of pretreatment of GW-39 stock tumors (10% GW-39
cell suspension) with interferon-γ (IFNγ). The experiments
involving interferon-gamma enhancing the antitumor effects of naked CEA
antibody (hMN-15) are conducted as follows.

[0276] GW-39 human colon cancer is grown subcutaneously in a mouse that
receives 100,000 units of IFN-gamma twice a day for 4 days. A control
mouse with GW-39 tumor is not given IFN. Experimental mice are injected
i.v. with a 5% suspension of GW-39 (w/v) from either of the two mice
(i.e., with or without IFN treatment) into two groups of eight. Four of
each receive tumor from the IFN-treated mice and four from the untreated
mice. One group of 8 mice then receive hMN-15 (100 μg per day×14
days and then twice weekly thereafter until experiment is ended), another
group receive CPT-11 at 160 μg/day×5 days (=20% of maximum
tolerated dose), a third group receives the same doses of antibody+drug
combined, and a fourth group is not treated at all. Animal weights are
measured and survival determined weekly. Also, samples of stock tumor
treated with IFN in the mice that are later implanted are also processed
for immunohistology to assess increase in CEA expression in the tumors
from mice treated with IFN-gamma, and this is controlled by also treating
the suspensions by immunohistology with an irrelvant IgG, such as Ag8,
which shows no CEA staining.

[0277] It is observed that IFNγ pretreatment increases the
expression of CEA in GW-39 tumors. The combination of naked hMN-15 with
CPT-11 is more effective at reducing tumor growth and increasing survival
than the sum of the effects of either therapeutic agent alone. This
effect is more pronounced in the tumors pretreated with IFNγ.

Example 6

Sigmoid Colon Cancer Therapy with Anti-CEA Antibody and GM-CSF

[0278] JR is a 62-year-old man who is refractive to chemotherapy with
5-fluorouracil and leucovorin to reduce his metastases to the liver found
at the time of removal of his sigmoid colon cancer. His plasma titer of
carcinoembryonic antigen (CEA) at presentation is 34 ng/mL, and computed
tomography of the liver shows several small lesions measuring between 2
and 4 cm in diameter in the right lobe. Other radiological studies appear
to be normal. Immunotherapy with humanized anti-CEA IgG (hMN-15)
monoclonal antibody is begun on a weekly basis for 4 weeks, at an
intravenous dose of 300 mg/m2 infused over 2 hours. One week prior
to hMN-15 therapy, the patient receives 2 subcutaneous injections of 200
μg/m2 GM-CSF, 3 days apart, and continued twice weekly during the
4 weeks of hMN-15 therapy. After these four weeks, both hMN-15 and GM-CSF
are given at the same doses every other week for an additional 3 months,
but the dose of GM-CSF is increased to 250 μg/m2. Prior to each
administration of the humanized anti-CEA antibody, the patient is given
diphenhydramine (50 mg orally), and acetaminophen (500 mg orally).

[0279] At this time, the patient is restaged, with CT measurements made of
the liver metastases and diverse radiological scans of the rest of the
body. Blood is also taken for chemistries and for determination of his
blood CEA titer. No areas of disease outside of the liver are noted, but
the sum of the diameters of the measurable tumors in the liver appear to
decrease by 40 percent, and the patient's blood CEA titer decreases to 18
ng/mL, thus indicating a therapeutic response.

[0280] Immunotherapy with hMN-15 and GM-CSF, given once every other week
at 200 mg/m2 for hMN-15 and 250 μg/m2 for GM-CSF, are
administered for another 2 months, and restaging shows additional
decrease in the sum of the diameters of the liver tumors and a fall in
the CEA titer to 10 ng/mL. Since tumor decrease is measured as being
>65% over the pre-therapy baseline, the therapy is considered to have
provided a partial response. After this, the doses are made less
frequent, once every month for the next six months, and all studies
indicate no change in disease. The patient is then followed for another
10 months, and remains in a partial remission, with no adverse reactions
to the therapy, and generally without any symptoms of disease.

Example 7

Combined Immunotherapy and Chemotherapy of Metastatic Colon Cancer

[0281] ST is a 52-year-old woman presenting with liver and lung metastases
of colon cancer following resection of the primary tumor. She is placed
on a combined chemotherapy and immunotherapy protocol based on the
Gramont schedule (de Gramont et al., J Clin Oncol. 2000, 18:2938-47), but
with the addition of humanized anti-CEA monoclonal antibody IgG1
(hMN-15). Prior to infusions of the antibody, she receives 50 mg orally
of diphenhydramine and 500 mg orally of acetaminophen. She receives a
2-hr infusion of leucovorin (200 mg/m2/day) followed by a bolus of
5-fluorouracil (400 mg/m2/day) and 22-hour continuous infusion of
5-fluorouracil (600 mg/m2/day) for 2 consecutive days every 2 weeks,
together with oxaliplatin at 85 mg/m2 as a 2-hr infusion in 250 mL
of dextrose 5%, concurrent with leukovorin on day 1 (FOLFOX4 schedule).
The patient also receives anti-emetic prophylaxis with a
5-hydroxyltryptamine-3-receptor antagonist. One week prior to this 2-week
chemotherapy cycle, hMN-15 monoclonal anti-CEA antibody is infused over 2
hrs at a dose of 200 mg/m2, and repeated each week of the 2-week
chemotherapy cycle, and every week thereafter for the next month with
another chemotherapy cycle. Also, a subcutaneous dose of 5 μg/kg/day
of G-CSF is administered once weekly beginning with the second
chemotherapy cycle, and continued at this dose for the duration of
immunotherapy with hMN-15 antibody, over the next 3 months.

[0282] A total of 5 cycles of chemotherapy is performed with continued
administration of hMN-15 antibody and filgrastim. Thereafter, hMN-15 and
filgrastim therapy is given, at the same doses, every other week for the
next 3 months, without chemotherapy. The patient is staged 2 months
later, and her liver and lung metastases show shrinkage by computed
tomography measurements of >80 percent of disease measured in the
liver and lungs, as compared to the measurements made prior to therapy.
Her blood CEA titer also shows a drop from the pre-therapy level of 63
ng/mL to 9 ng/mL. She is followed over the next 6 months, and her disease
appears to be stable, with no new lesions found and no increase in the
disease remaining in the liver and lungs. The patient's predominant
toxicity is peripheral sensory neuropathy, which consists of
laryngeopharyngeal dysesthesia. The patient also experiences diarrhea,
mucositis, nausea and vomiting during the chemotherapy cycles, but these
are not excessive. She does not experience any adverse events when only
immunotherapy is administered, and is able to return to full-time
activities without any significant restrictions.

Example 8

Effect of hMN-15 Pretreatment on CPT-11 Efficacy

[0283] The effects of pre-treatment with naked hMN-15 CEA Mab given 3 days
prior to CPT-11 treatment is examined in a SUM1315 breast cancer model
(Kuperwasser et al., Cancer Res. 2005, 65:6130-38). CPT-11 alone, hMN-15
alone, and combination therapy of hMN-15+CPT-11 where the hMN-15 is
administered 3 days prior to the CPT-11 are compared. Dosages are as
indicated in Example 5. hMN-15 alone increases median survival time by
21% under these conditions. CPT 11 alone increases survival by 76%. By
contrast, the combination therapy where hMN-15 is administered 3 days
prior to CPT-11 produces a median survival time increase of an additional
58% above CPT-11 alone. Pre-treatment with hMN-15 significantly prolongs
survival of animals with low tumor burden in a metastatic model of human
breast cancer. The synergistic effect of hMN-15 antibody with CPT-11
therapy is surprising.

Example 9

Effect of Administration Schedule on hMN-15 Synergy with CPT-11

[0284] A comparison is made of various administration schedules of naked
hMN-15 CEA Mab and CPT-11 in a human colon cancer model. Giving hMN-15 3
days before CPT-11 is the most effective. Dosages are as indicated
Example 5. When the order is reversed (CPT-11 is given 3 days before
hMN-15) or when both are given together at the same time, median survival
time of 70 days is an increase over the untreated control group (35 days)
but is still less than the median survival time of 105 days with the
hMN-15 pre-treatment 3 days before CPT-11.

[0286] Tumors are propagated in female nu/nu mice (Taconic Farms,
Germantown, N.Y.) at 6-8 weeks of age by s.c. injection of
2×108 washed TT cells, which is propagated in tissue culture.
Antibodies are injected i.v., via the lateral tail vein, into the
tumor-bearing animals. Tumor size is monitored by weekly measurements of
the length, width, and depth of the tumor using a caliper. Tumor volume
is calculated as the product of the three measurements.

[0287] To study whether naked hMN-15 can add to the efficacy of DTIC, TT
bearing nude mice are given DTIC (75 μg/dose) in combination with a
course of treatment of the unlabeled MAb. DTIC is administered for 3
consecutive days at 75 μg/dose as one course, beginning 2 days after
s.c. injection of TT cells. hMN-15 MAb treatment is initiated 3 days
prior to the first dose of DTIC, at 100 μg/dose/day for 5 days in the
first two weeks, then twice weekly. Significant delays in tumor growth
are caused by these schedules of either MAb therapy or chemotherapy
alone. Surprisingly, the 75-μg dose of DTIC in combination with this
schedule of hMN-15 is more effective than either treatment alone. At 7
weeks, 8/10 mice in the 75 μg DTIC+MAb group have no palpable tumor,
compared to 1/10 in the 75 μg DTIC-only group and 0/10 in the
untreated and MAb-only groups. Mean tumor volumes at 7 weeks are
0.018+0.039 cm3 (75 μg DTIC+hMN-15), 0.284+0.197 cm3 (75
μg DTIC), 0.899+0.545 cm3 (hMN-15) and 1.578+0.959 cm3
(untreated).

Example 11

Preparation of DNL Constructs for Pretargeting

[0288] In various forms, the DNL technique may be used to make dimers,
trimers, tetramers, hexamers, etc. comprising virtually any antibodies or
fragments thereof or other effector moieties, such as cytokines. For
certain preferred embodiments, IgG antibodies or Fab antibody fragments
may be produced as fusion proteins containing either a DDD or AD
sequence. Bispecific antibodies may be formed by combining a Fab-DDD
fusion protein of a first antibody with a Fab-AD fusion protein of a
second antibody. Alternatively, constructs may be made that combine
IgG-AD fusion proteins with Fab-DDD fusion proteins. For purposes of
pre-targeting, an antibody or fragment containing a binding site for an
antigen associated with a target tissue to be treated, such as a tumor,
may be combined with a second antibody or fragment that binds a hapten on
a targetable construct. In exemplary embodiments, the tumor targeting
antibody or fragment is a chimeric, humanized or human MN-15 antibody
comprising the 6 MN-15 CDR sequences, while the hapten binding moiety may
be an anti-HSG or anti-DTPA antibody. The bispecific antibody (DNL
construct) is administered to a subject, circulating antibody is allowed
to clear from the blood and localize to target tissue, and a targetable
construct attached to at least one therapeutic and/or diagnostic agent is
added and binds to the localized antibody.

[0289] Independent transgenic cell lines may be developed for each Fab or
IgG fusion protein. Once produced, the modules can be purified if desired
or maintained in the cell culture supernatant fluid. Following
production, any DDD2-fusion protein module can be combined with any
AD-fusion protein module to generate a bispecific DNL construct. For
different types of constructs, different AD or DDD sequences may be
utilized.

[0290] The plasmid vector pdHL2 has been used to produce a number of
antibodies and antibody-based constructs. (See, Gillies et al., J Immunol
Methods (1989), 125:191-202; Losman et al., Cancer (Phila) (1997),
80:2660-6.) The di-cistronic mammalian expression vector directs the
synthesis of the heavy and light chains of IgG. The vector sequences are
mostly identical for many different IgG-pdHL2 constructs, with the only
differences existing in the variable domain (VH and VL) sequences. Using
molecular biology tools known to those skilled in the art, these IgG
expression vectors can be converted into Fab-DDD or Fab-AD expression
vectors. To generate Fab-DDD expression vectors, the coding sequences for
the hinge, CH2 and CH3 domains of the heavy chain are replaced with a
sequence encoding the first 4 residues of the hinge, a 14 residue Gly-Ser
linker and the first 44 residues of human RIIα (referred to as
DDD1, SEQ ID NO:11). To generate Fab-AD expression vectors, the sequences
for the hinge, CH2 and CH3 domains of IgG are replaced with a
sequence encoding the first 4 residues of the hinge, a 15 residue Gly-Ser
linker and a 17 residue synthetic AD called AKAP-IS (referred to as AD1,
SEQ ID NO:13), which was generated using bioinformatics and peptide array
technology and shown to bind RIIα dimers with a very high affinity
(0.4 nM). See Alto, et al. Proc. Natl. Acad. Sci., U.S.A (2003),
100:4445-50.

[0291] Two shuttle vectors were designed to facilitate the conversion of
IgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, as
described below.

[0292] Preparation of CH1

[0293] The CH1 domain was amplified by PCR using the pdHL2 plasmid vector
as a template. The left PCR primer consisted of the upstream (5') end of
the CH1 domain and a SacII restriction endonuclease site, which is 5' of
the CH1 coding sequence. The right primer consisted of the sequence
coding for the first 4 residues of the hinge (PKSC) followed by four
glycines and a serine, with the final two codons (GS) comprising a Bam HI
restriction site. The 410 by PCR amplimer was cloned into the pGemT PCR
cloning vector (Promega, Inc.) and clones were screened for inserts in
the T7 (5') orientation.

[0294] Construction of (G4S)2DDD1 ((G4S)2 Disclosed as
SEQ ID NO: 15)

[0295] A duplex oligonucleotide, designated (G4S)2DDD1
((G4S)2 disclosed as SEQ ID NO: 15), was synthesized by Sigma
Genosys (Haverhill, UK) to code for the amino acid sequence of DDD1
preceded by 11 residues of the linker peptide, with the first two codons
comprising a BamHI restriction site. A stop codon and an EagI restriction
site are appended to the 3' end. The encoded polypeptide sequence is
shown below.

[0296] Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom,
that overlap by 30 base pairs on their 3' ends, were synthesized (Sigma
Genosys) and combined to comprise the central 154 base pairs of the 174
by DDD1 sequence. The oligonucleotides were annealed and subjected to a
primer extension reaction with Taq polymerase. Following primer
extension, the duplex was amplified by PCR. The amplimer was cloned into
pGemT and screened for inserts in the T7 (5') orientation.

[0297] Construction of (G4S)2-AD1 ((G45)2 Disclosed as
SEQ ID NO: 15)

[0298] A duplex oligonucleotide, designated (G4S)-2-AD1
((G45)2 disclosed as SEQ ID NO: 15), was synthesized (Sigma
Genosys) to code for the amino acid sequence of AD1 preceded by 11
residues of the linker peptide with the first two codons comprising a
BamHI restriction site. A stop codon and an EagI restriction site are
appended to the 3' end. The encoded polypeptide sequence is shown below.

TABLE-US-00004
(SEQ ID NO: 17)
GSGGGGSGGGGSQIEYLAKQIVDNAIQQA

[0299] Two complimentary overlapping oligonucleotides encoding the above
peptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, were
synthesized and annealed. The duplex was amplified by PCR. The amplimer
was cloned into the pGemT vector and screened for inserts in the T7 (5')
orientation.

[0300] Ligating DDD1 with CH1

[0301] A 190 by fragment encoding the DDD1 sequence was excised from pGemT
with BamHI and NotI restriction enzymes and then ligated into the same
sites in CH1-pGemT to generate the shuttle vector CH1-DDD1-pGemT.

[0302] Ligating AD1 with CH1

[0303] A 110 by fragment containing the AD1 sequence was excised from
pGemT with BamHI and NotI and then ligated into the same sites in
CH1-pGemT to generate the shuttle vector CH1-AD1-pGemT.

[0304] Cloning CH1-DDD1 or CH1-AD1 into pdHL2-based vectors

[0305] With this modular design either CH1-DDD1 or CH1-AD1 can be
incorporated into any IgG construct in the pdHL2 vector. The entire heavy
chain constant domain is replaced with one of the above constructs by
removing the SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and
replacing it with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which
is excised from the respective pGemT shuttle vector.

[0306] Construction of h679-Fd-AD1-pdHL2

[0307] h679-Fd-AD1-pdHL2 is an expression vector for production of h679
Fab with AD1 coupled to the carboxyl terminal end of the CH1 domain of
the Fd via a flexible Gly/Ser peptide spacer composed of 14 amino acid
residues. A pdHL2-based vector containing the variable domains of h679
was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagI
fragment with the CH1-AD1 fragment, which was excised from the
CH1-AD1-SV3 shuttle vector with SacII and EagI.

[0308] Construction of C-DDD1-Fd-hMN-15-pdHL2

[0309] C-DDD1-Fd-hMN-15-pdHL2 is an expression vector for production of a
stable dimer that comprises two copies of a fusion protein
C-DDD1-Fab-hMN-15, in which DDD1 is linked to hMN-15 Fab at the carboxyl
terminus of CH1 via a flexible peptide spacer. The plasmid vector
hMN15-pdHL2, which is used to produce hMN-15 IgG, is converted to
C-DDD1-Fd-hMN-15-pdHL2 by digestion with SacII and EagI restriction
endonucleases to remove the CH1-CH3 domains and insertion of the CH1-DDD1
fragment, which is excised from the CH1-DDD1-SV3 shuttle vector with
SacII and EagI.

[0310] The same technique has been utilized to produce plasmids for Fab
expression of a wide variety of known antibodies, such as hLL1, hLL2,
hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others. Generally,
the antibody variable region coding sequences were present in a pdHL2
expression vector and the expression vector was converted for production
of an AD- or DDD-fusion protein as described above. The AD- and
DDD-fusion proteins comprising a Fab fragment of any of such antibodies
may be combined, in an approximate ratio of two DDD-fusion proteins per
one AD-fusion protein, to generate a trimeric DNL construct comprising
two Fab fragments of a first antibody and one Fab fragment of a second
antibody.

[0311] C-DDD2-Fd-hMN-15-pdHL2

[0312] C-DDD2-Fd-hMN-15-pdHL2 is an expression vector for production of
C-DDD2-Fab-hMN-15, which possesses a dimerization and docking domain
sequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-15
via a 14 amino acid residue Gly/Ser peptide linker. The fusion protein
secreted is composed of two identical copies of hMN-15 Fab held together
by non-covalent interaction of the DDD2 domains.

[0313] The expression vector is engineered as follows. Two overlapping,
complimentary oligonucleotides, which comprise the coding sequence for
part of the linker peptide (GGGGSGGGCG, SEQ ID NO:18) and residues 1-13
of DDD2 (SEQ ID NO:12), are made synthetically. The oligonucleotides are
annealed and phosphorylated with T4 PNK, resulting in overhangs on the 5'
and 3' ends that are compatible for ligation with DNA digested with the
restriction endonucleases BamHI and PstI, respectively.

[0314] The duplex DNA is ligated with the shuttle vector CH1-DDD1-pGemT,
which is prepared by digestion with BamHI and PstI, to generate the
shuttle vector CH1-DDD2-pGemT. A 507 by fragment is excised from
CH1-DDD2-pGemT with SacII and EagI and ligated with the IgG expression
vector hMN15-pdHL2, which is prepared by digestion with SacII and EagI.
The final expression construct is designated C-DDD2-Fd-hMN-15-pdHL2.
Similar techniques have been utilized to generated DDD2-fusion proteins
of the Fab fragments of a number of different humanized antibodies.

[0315] H679-Fd-AD2-pdHL2

[0316] h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-15 as A.
h679-Fd-AD2-pdHL2 is an expression vector for the production of
h679-Fab-AD2, which possesses an anchor domain sequence of AD2 (SEQ ID
NO:14) appended to the carboxyl terminal end of the CH1 domain via a 14
amino acid residue Gly/Ser peptide linker. AD2 has one cysteine residue
preceding and another one following the anchor domain sequence of AD1.

[0317] The expression vector was engineered as follows. Two overlapping,
complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprise
the coding sequence for AD2 and part of the linker sequence, were made
synthetically. The oligonucleotides were annealed and phosphorylated with
T4 PNK, resulting in overhangs on the 5' and 3' ends that are compatible
for ligation with DNA digested with the restriction endonucleases BamHI
and SpeI, respectively.

[0318] The duplex DNA was ligated into the shuttle vector CH1-AD1-pGemT,
which was prepared by digestion with BamHI and SpeI, to generate the
shuttle vector CH1-AD2-pGemT. A 429 base pair fragment containing CH1 and
AD2 coding sequences was excised from the shuttle vector with SacII and
EagI restriction enzymes and ligated into h679-pdHL2 vector that prepared
by digestion with those same enzymes. The final expression vector is
h679-Fd-AD2-pdHL2.

[0319] Generation of Trimeric MN-15-679 Construct

[0320] A trimeric DNL construct is obtained by reacting C-DDD2-Fab-hMN-15
with h679-Fab-AD2. A pilot batch of DNL trimer is generated with >90%
yield as follows. Protein L-purified C-DDD2-Fab-hMN-15 (200 mg) is mixed
with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. The total protein
concentration is 1.5 mg/ml in PBS containing 1 mM EDTA. Subsequent steps
involved TCEP reduction, HIC chromatography, DMSO oxidation, and IMP 291
affinity chromatography. Addition of 5 mM TCEP rapidly results in the
formation of a2b complex consistent with a 157 kDa protein expected
for the binary structure. The trimeric DNL construct is purified to near
homogeneity by IMP 291 affinity chromatography. IMP 291 is a synthetic
peptide containing the HSG hapten to which the 679 Fab binds (Rossi et
al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLC analysis of the IMP 291
unbound fraction demonstrates the removal of a4, a2 and free
kappa chains from the product. Binding studies indicate that the trimeric
DNL construct incorporating the hMN-15 antibody binds to CEA and HSG,
with affinities similar to the parent MN-15 and 679 antibodies.

[0321] Production of TF10 Bispecific Antibody

[0322] A similar protocol was used to generate a trimeric TF10 DNL
construct, comprising two copies of a C-DDD2-Fab-hPAM4 and one copy of
C-AD2-Fab-679. The cancer-targeting antibody component in TF10 is derived
from hPAM4, a humanized anti-pancreatic cancer mucin MAb that has been
studied in detail as a radiolabeled MAb (e.g., Gold et al., Clin. Cancer
Res. 13: 7380-7387, 2007). The hapten-binding component is derived from
h679, a humanized anti-histaminyl-succinyl-glycine (HSG) MAb discussed
above. The TF10 bispecific ([hPAM4]2×h679) antibody was
produced using the method disclosed for production of the (anti
CEA)2× anti HSG bsAb, as described above. The TF10 construct
bears two humanized PAM4 Fabs and one humanized 679 Fab.

[0323] The two fusion proteins (hPAM4-DDD and h679-AD2) were expressed
independently in stably transfected myeloma cells. The tissue culture
supernatant fluids were combined, resulting in a two-fold molar excess of
hPAM4-DDD. The reaction mixture was incubated at room temperature for 24
hours under mild reducing conditions using 1 mM reduced glutathione.
Following reduction, the DNL reaction was completed by mild oxidation
using 2 mM oxidized glutathione. TF10 was isolated by affinity
chromatography using IMP 291-affigel resin, which binds with high
specificity to the h679 Fab.

[0324] hMN-15-Fd-AD2-pdHL2

[0325] For certain DNL constructs, it is preferred to use an
hMN-15-Fab-AD2 fusion protein, which possesses an anchor domain sequence
of AD2 (SEQ ID NO:14) appended to the carboxyl terminal end of the CH1
domain via a 14 amino acid residue Gly/Ser peptide linker. An
hMN-15-Fab-AD2 construct may be utilized, for example, to prepare a DNL
construct comprising a lower number of hMN-15 subunits and a greater
number of other effector moiety subunits, such as a toxin, cytokine, drug
or another antibody or antibody fragment.

[0326] The expression vector is engineered as described above for the
h679-Fab-AD2 fusion protein. Two overlapping, complimentary
oligonucleotides (AD2 Top and AD2 Bottom), which comprise the coding
sequence for AD2 and part of the linker sequence, are made synthetically.
The oligonucleotides are annealed and phosphorylated with T4 PNK,
resulting in overhangs on the 5' and 3' ends that are compatible for
ligation with DNA digested with the restriction endonucleases BamHI and
SpeI, respectively.

[0327] The duplex DNA is ligated into the shuttle vector CH1-AD1-pGemT,
which is prepared by digestion with BamHI and SpeI, to generate the
shuttle vector CH1-AD2-pGemT. A 429 base pair fragment containing CH1 and
AD2 coding sequences is excised from the shuttle vector with SacII and
EagI restriction enzymes and ligated into hMN-15-pdHL2 vector prepared by
digestion with those same enzymes. The final expression vector is
hMN-15-Fd-AD2-pdHL2.

Example 12

Imaging Using Pretargeting with hMN-15-679 DNL Construct and
111In-Labeled Peptides

[0328] The following study demonstrates the feasibility of in vivo imaging
using the pretargeting technique with labeled targeting peptides and
bispecific antibodies incorporating hMN-15. The hMN-15-679 DNL construct,
comprising two copies of a C-DDD2-Fab-hMN-15 and one copy of
C-AD2-Fab-679, is prepared as described in the preceding Example. Nude
mice bearing 0.2 to 0.3 g human colon cancer xenografts are imaged, using
pretargeting with the hMN-15-679 DNL construct and an 111In-IMP-288
peptide. The results show clearly delineated tumors in animal models
using a bsMAb pretargeting method. Experimental animals receive different
doses of hMN-15-679 DNL construct at 10:1 or 20:1 mole ratio to the moles
of peptide given, and the next day they are given an 1111n-labeled
diHSG peptide (IMP 288). The control animals receive only the
111In-IMP-288 (no pretargeting). The images are taken 3 h after the
injection of the labeled peptide and show clear localization of 0.2-0.3 g
tumors in the pretargeted animals with both doses of DNL construct, with
no localization in the animals given the 111In-peptide alone.

Example 13

PEGylated DNL Constructs

[0329] In certain embodiments, it may be preferred to prepare constructs
comprising PEGylated forms of antibody or immunoconjugate, for example to
increase the serum half-life of the antibody or immunoconjugate moiety.
Such PEGylated constructs may be prepared by the DNL technique.

[0330] In a preferred method, the effector moiety to be PEGylated, such as
hMN-15 Fab, is linked to a DDD sequence to generate the DDD module. A PEG
reagent of a selected molecular size is derivatized with a complementary
AD sequence and the resulting PEG-AD module is combined with the DDD
module to produce a PEGylated conjugate that consists of a single PEG
tethered site-specifically to two copies of the hMN-15 Fab or other
effector moiety via the disulfide bonds formed between DDD and AD. The
PEG reagents may be capped at one end with a methoxy group (m-PEG), can
be linear or branched, and may contain one of the following functional
groups: propionic aldehyde, butyric aldehyde, ortho-pyridylthioester
(OPTE), N-hydroxysuccinimide (NHS), thiazolidine-2-thione, succinimidyl
carbonate (SC), maleimide, or ortho-pyridyldisulfide (OPPS). Among the
effector moieties that may be of interest for PEGylation are enzymes,
cytokines, chemokines, growth factors, peptides, aptamers, hemoglobins,
antibodies and antibody fragments. The method is not limiting and a wide
variety of agents may be PEGylated using the disclosed methods and
compositions. PEG of various sizes and derivatized with a variety of
reactive moieties may be obtained from commercial sources, such as
NEKTAR® Therapeutics (Huntsville, Ala.).

[0332] IMP350, incorporating the sequence of AD2, was made on a 0.1 mmol
scale with Sieber Amide resin using Fmoc methodology on a peptide
synthesizer. Starting from the C-terminus the protected amino acids used
were Fmoc-Cys(t-Buthio)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Asp(OBut)-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Tyr(But)-OH,
Fmoc-Glu(OBut)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH and
Fmoc-Cys(Trt)-OH. The peptide was cleaved from the resin and purified by
reverse phase (RP)-HPLC.

[0333] Synthesis of PEG20-IMP350

[0334] IMP350 (0.0104 g) was mixed with 0.1022 g of mPEG-OPTE (20 kDa,
NEKTAR® Therapeutics) in 7 mL of 1 M Tris buffer at pH 7.81.
Acetonitrile, 1 mL, was then added to dissolve some suspended material.
The reaction was stirred at room temperature for 3 h and then 0.0527 g of
TCEP was added along with 0.0549 g of cysteine. The reaction mixture was
stirred for 1.5 h and then purified on a PD-10 desalting column, which
was equilibrated with 20% methanol in water. The sample was eluted,
frozen and lyophilized to obtain 0.0924 g of crude PEG20-IMP350
(MH+23508 by MALDI).

[0336] IMP 360, incorporating the AD2 sequence, was synthesized on a 0.1
mmol scale with Fmoc-Gly-EDANS resin using Fmoc methodology on a peptide
synthesizer. The Fmoc-Gly-OH was added to the resin manually using 0.23 g
of Fmoc-Gly-OH, 0.29 g of HATU, 26 μL of DIEA, 7.5 mL of DMF and 0.57
g of EDANS resin (NOVABIOCHEM®). The reagents were mixed and added to
the resin. The reaction was mixed at room temperature for 2.5 hr and the
resin was washed with DMF and IPA to remove the excess reagents. Starting
from the C-terminus the protected amino acids used were
Fmoc-Cys(t-Buthio)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH,
Fmoc-Asp(OBut)-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Tyr(But)-OH,
Fmoc-Glu(OBut)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH and
Fmoc-Cys(Trt)-OH. The peptide was cleaved from the resin and purified by
RP-HPLC.

[0337] For synthesis of IMP362, IMP360 (0.0115 g) was mixed with 0.1272 g
of mPEG-OPTE (20 kDa, NEKTAR® Therapeutics) in 7 mL of 1 M tris
buffer, pH 7.81. Acetonitrile (1 mL) was then added to dissolve some
suspended material. The reaction was stirred at room temperature for 4 h
and then 0.0410 g of TCEP was added along with 0.0431 g of cysteine. The
reaction mixture was stirred for 1 h and purified on a PD-10 desalting
column, which was equilibrated with 20% methanol in water. The sample was
eluted, frozen and lyophilized to obtain 0.1471 g of crude IMP362
(MH+23713).

[0338] Synthesis of IMP413 (PEG30-IMP360)

[0339] For synthesis of IMP 413, IMP 360 (0.0103 g) was mixed with 0.1601
g of mPEG-OPTE (30 kDa, NEKTAR® Therapeutics) in 7 mL of 1 M tris
buffer at pH 7.81. Acetonitrile (1 mL) was then added to dissolve some
suspended material. The reaction was stirred at room temperature for 4.5
h and then 0.0423 g of TCEP was added along with 0.0473 g of cysteine.
The reaction mixture was stirred for 2 h followed by dialysis for two
days. The dialyzed material was frozen and lyophilized to obtain 0.1552 g
of crude IMP413 (MH.sup.+ 34499).

[0341] The peptide IMP421, MH.sup.+ 2891 was made on NOVASYN® TGR
resin (487.6 mg, 0.112 mmol) by adding the following amino acids to the
resin in the order shown: Fmoc-Gly-OH, Fmoc-Cys(t-Buthio)-OH,
Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OBut)-OH,
Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Tyr(But)-OH, Fmoc-Glu(OBut)-OH,
Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Cys(t-Buthio)-OH,
Fmoc-NH-PEG3-COOH, Fmoc-Cys(Trt)-OH. The N-terminal amino acid was
protected as an acetyl derivative. The peptide was then cleaved from the
resin and purified by RP-HPLC to yield 32.7 mg of a white solid.

[0342] Synthesis of IMP457

[0343] IMP 421 (SEQ ID NO:21), incorporating the sequence of AD2, was
synthesized by standard chemical means. To a solution of 15.2 mg (5.26
μmol) IMP 421 (F.W. 2890.50) and 274.5 mg (6.86 μmol) mPEG2-MAL-40K
in 1 mL of acetonitrile was added 7 mL 1 M Tris pH 7.8 and allowed to
react at room temperature for 3 h. The excess mPEG2-MAL-40K was quenched
with 49.4 mg L-cysteine, followed by S--S-tBu deprotection over one hour
with 59.1 mg TCEP. The reaction mixture was dialyzed overnight at
2-8° C. using two 3-12 mL capacity 10K SLIDE-A-LYZER® dialysis
cassettes (4 ml into each cassette) into 5 L of 5 mM ammonium acetate, pH
5.0. Three more 5 L buffer changes of 5 mM ammonium acetate, pH 5.0 were
made the next day with each dialysis lasting at least 21/2 h. The
purified product (19.4 mL) was transferred into two 20 mL scintillation
vials, frozen and lyophilized to yield 246.7 mg of a white solid.
MALDI-TOF gave results of mPEG2-MAL-40K 42,982 and IMP-457 45,500.

Example 14

Generation of PEGylated hMN-15 by DNL

[0344] A DNL structure is prepared having two copies of hMN-15 Fab coupled
to a 20 kDa PEG. A DNL reaction is performed by the addition of reduced
and lyophilized IMP362 in 10-fold molar excess to hMN-15 Fab-DDD2 in 250
mM imidazole, 0.02% Tween 20, 150 mM NaCl, 1 mM EDTA, 50 mM
NaH2PO4, pH 7.5. After 6 h at room temperature in the dark, the
reaction mixture is dialyzed against CM Loading Buffer (150 mM NaCl, 20
mM NaAc, pH 4.5) at 4° C. in the dark. The solution is loaded onto
a 1-mL Hi-Trap CM-FF column (AMERSHAM®), which is pre-equilibrated
with CM Loading buffer. After sample loading, the column is washed with
CM loading buffer to baseline, followed by washing with 15 mL of 0.25 M
NaCl, 20 mM NaAc, pH 4.5. The PEGylated hMN-15 is eluted with 12.5 mL of
0.5 M NaCl, 20 mM NaAc, pH 4.5.

[0345] The conjugation process is analyzed by SDS-PAGE with Coomassie blue
staining. Under non-reducing conditions, the Coomassie blue-stained gel
reveals the presence of a major band in the reaction mixture, which is
absent in the unbound or 0.25 M NaCl wash fraction, but evident in the
0.5 M NaCl fraction. Fluorescence imaging, which is used to detect the
EDANS tag on IMP362, demonstrates that the band contains IMP362 and the
presence of excess IMP362 in the reaction mixture and the unbound
fraction. The DNL reaction results in the site-specific and covalent
conjugation of IMP362 with a dimer of hMN-15 Fab. Under reducing
conditions, which breaks the disulfide linkage, the components of the DNL
structures are resolved. The calculated MW of the (hMN-15 Fab)2-PEG
construct matches that determined by MALDI TOF. Overall, the DNL reaction
results in a near quantitative yield of a homogeneous product that is
>90% pure after purification by cation-exchange chromatography.

[0346] Another DNL reaction is performed by the addition of reduced and
lyophilized 1 MP457 in 10-fold molar excess to hMN-15 Fab-DDD2 in 250 mM
imidazole, 0.02% Tween 20, 150 mM NaCl, 1 mM EDTA, 50 mM
NaH2PO4, pH 7.5. After 60 h at room temperature, 1 mM oxidized
glutathione is added to the reaction mixture, which is then held for an
additional 2 h. The mixture is diluted 1:20 with CM Loading Buffer (150
mM NaCl, 20 mM NaAc, pH 4.5) and titrated to pH 4.5 with acetic acid. The
solution is loaded onto a 1-mL Hi-Trap CM-FF column (AMERSHAM®),
which is pre-equilibrated with CM Loading Buffer. After sample loading,
the column is washed with CM Loading Buffer to baseline, followed by
washing with 15 mL of 0.25 M NaCl, 20 mM NaAc, pH 4.5. The PEGylated
product is eluted with 20 mL of 0.5 M NaCl, 20 mM NaAc, pH 4.5. The DNL
construct is concentrated to 2 mL and diafiltered into 0.4 M PBS, pH 7.4.
The final PEGylated hMN-15 Fab2 construct is approximately 90%
purity as determined by SDS-PAGE.

[0347] A DNL construct having two copies of hMN-15 Fab coupled to a 30 kDa
PEG is prepared as described immediately above using IMP413 instead of
IMP362. The PEGylated hMN-15 Fab2 DNL construct is purified as
described above and obtained in approximately 90% purity. The PEGylated
DNL constructs may be used for therapeutic methods as described above for
non-PEGylated forms of hMN-15. It is observed that PEGylation of hMN-15
Fab2 increases the serum half-life of the hMN-15 moiety, as
expected.

Example 15

Generation of DDD Module Based on Interferon (IFN)-α2b

[0348] The cDNA sequence for IFN-α2b was amplified by PCR, resulting
in a sequence comprising the following features, in which XbaI and BamHI
are restriction sites, the signal peptide is native to IFN-α2b, and
6 His is a hexahistidine tag (SEQ ID NO:37): XbaI--Signal
peptide--IFNα2b--6 His--BamHI ("6 His" disclosed as SEQ ID NO:37).
The resulting secreted protein consists of IFN-α2b fused at its
C-terminus to a polypeptide consisting of SEQ ID NO:22.

[0349] PCR amplification was accomplished using a full length human
IFNα2b cDNA clone (INVITROGEN® Ultimate ORF human clone
cat#HORF01Clone ID IOH35221) as a template and the following
oligonucleotides as primers:

[0350] The PCR amplimer was cloned into the PGEMT® vector
(PROMEGA®). A DDD2-pdHL2 mammalian expression vector was prepared for
ligation with IFN-α2b by digestion with XbaI and Bam HI restriction
endonucleases. The IFN-α2b amplimer was excised from PGEMT®
with XbaI and Bam HI and ligated into the DDD2-pdHL2 vector to generate
the expression vector IFN-α2b-DDD2-pdHL2.

[0351] IFN-α2b-DDD2-pdHL2 was linearized by digestion with Sail
enzyme and stably transfected into Sp/EEE myeloma cells by
electroporation (see, e.g., U.S. Pat. No. 7,537,930, the Examples section
of which is incorporated herein by reference). Two clones were found to
have detectable levels of IFN-α2b by ELISA. One of the two clones,
designated 95, was adapted to growth in serum-free media without
substantial decrease in productivity. The clone was subsequently
amplified with increasing methotrexate (MTX) concentrations from 0.1 to
0.8 μM over five weeks. At this stage, it was sub-cloned by limiting
dilution and the highest producing sub-clone (95-5) was expanded. The
productivity of 95-5 grown in shake-flasks was estimated to be 2.5 mg/L
using commercial rIFN-α2b (CHEMICON® IF007, Lot 06008039084) as
a standard.

[0353] The purity of IFN-α2b-DDD2 was assessed by SDS-PAGE under
reducing conditions (not shown). IFN-α2b-DDD2 was the most heavily
stained band and accounted for approximately 50% of the total protein
(not shown). The product resolved as a doublet with an Mr of
˜26 kDa, which is consistent with the calculated MW of
IFN-α2b-DDD2-SP (26 kDa). There was one major contaminant with a
Mr of 34 kDa and many faint contaminating bands (not shown).

Example 16

Generation of hMN-15 Fab-(IFN-α2b)2 by DNL

[0354] Creation of C--H-AD2-IgG-pdHL2 Expression Vectors.

[0355] The pdHL2 mammalian expression vector has been used to mediate the
expression of many recombinant IgGs. A plasmid shuttle vector was
produced to facilitate the conversion of any IgG-pdHL2 vector into a
C--H-AD2-IgG-pdHL2 vector. The gene for the Fc (CH2 and CH3 domains) was
amplified using the pdHL2 vector as a template and the oligonucleotides
Fc BglII Left and Fc Bam-EcoRI Right as primers.

[0356] The amplimer was cloned in the PGEMT® PCR cloning vector. The
Fc insert fragment was excised from PGEMT® and ligated with AD2-pdHL2
vector to generate the shuttle vector Fc-AD2-pdHL2.

[0357] Generation of hMN-15 IgG-AD2

[0358] To convert any IgG-pdHL2 expression vector to a C--H-AD2-IgG-pdHL2
expression vector, an 861 by BsrGI/NdeI restriction fragment is excised
from the former and replaced with a 952 by BsrGI/NdeI restriction
fragment excised from the Fc-AD2-pdHL2 vector. BsrGI cuts in the CH3
domain and NdeI cuts downstream (3') of the expression cassette. This
method is used to generate an hMN-15 IgG-AD2 protein.

[0359] Generation of hMN-15 IgG-(IFN-α2b)2 Construct

[0360] A DNL reaction is performed by the addition of reduced and
lyophilized hMN-15 IgG-AD2 to IFN-α2b-DDD2 in 250 mM imidazole,
0.02% Tween 20, 150 mM NaCl, 1 mM EDTA, 50 mM NaH2PO4, pH 7.5.
After 6 h at room temperature in the dark, the reaction mixture is
dialyzed against CM Loading Buffer (150 mM NaCl, 20 mM NaAc, pH 4.5) at
4° C. in the dark. The solution is loaded onto a 1-mL Hi-Trap
CM-FF column (AMERSHAM®), which is pre-equilibrated with CM Loading
buffer. After sample loading, the column is washed with CM loading buffer
to baseline, followed by washing with 15 mL of 0.25 M NaCl, 20 mM NaAc,
pH 4.5. The product is eluted with 12.5 mL of 0.5 M NaCl, 20 mM NaAc, pH
4.5. The DNL reaction results in the site-specific and covalent
conjugation of hMN-15 IgG with a dimer of IFN-α2b. Both the IgG and
IFN-α2b moieties retain their respective physiological activities
in the DNL construct. This technique may be used to attach any cytokine
or other physiologically active protein or peptide to hMN-15 for targeted
delivery to colon cancer or other cancers that express the CEA antigen.

Example 17

Preparation of DNL Bispecific Antibody Constructs

[0361] Methods of preparing bispecific DNL antibody constructs are
described, for example, in U.S. Pat. No. 7,521,056, the methods section
of which is incorporated herein by reference.

[0362] Construction of C-DDD1-Fd-hMN-14-pdHL2

[0363] C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of a
stable dimer that comprises two copies of a fusion protein
C-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxyl
terminus of CH1 via a flexible peptide spacer. The plasmid vector
hMN14(I)-pdHL2, which has been used to produce hMN-14 IgG, was converted
to C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagI restriction
endonucleases to remove the CH1-CH3 domains and insertion of the CH1-DDD1
fragment, which was excised from the CH1-DDD1-SV3 shuttle vector with
SacII and EagI.

[0364] Production of C-DDD2-Fd-hMN-14-pdHL2

[0365] C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production of
C-DDD2-Fab-hMN-14, which possesses a dimerization and docking domain
sequence of DDD2 appended to the carboxyl terminus of the Fd via a 14
amino acid residue Gly/Ser peptide linker. The fusion protein secreted is
composed of two identical copies of hMN-14 Fab held together by
non-covalent interaction of the DDD2 domains.

[0366] The C-DDD2-Fd-hMN-14-pdHL2 vector was transfected into Sp/EEE
myeloma cells by electroporation. The di-cistronic expression vector
directs the synthesis and secretion of both hMN-14 kappa light chain and
C-DDD2-Fd-hMN-14, which combine to form C-DDD2-Fab-hMN14. Dimerization
occurs via the DDD2 moiety, resulting in two reactive sulfhydryl groups
provided by the cysteine residue in each DDD2. Following electroporation,
the cells were plated in 96-well tissue culture plates and transfectant
clones were selected with 0.05 μM methotrexate (MTX).

[0367] Clones were screened for protein expression by ELISA using
microtitre plates coated with WI2 (hMN-14 anti-Id) and detection with
goat anti-human Fab-HRP. The highest producing clones had an initial
productivity of approximately 100 mg/L. A total of 200 mg of
C-DDD2-hMN-14 was purified by protein L affinity chromatography from 1.8
liters of roller bottle culture.

[0368] Generation of Trimeric hMN-14-hMN-15 Bispecific DNL Construct

[0369] hMN-15-Fab-AD2 is prepared as described in Example 12.
hMN-14-Fab-DDD2 is prepared as described above. The two fusion proteins
(hMN-14-Fab-DDD2 and hMN-15-Fab-AD2) are expressed independently in
stably transfected myeloma cells. The tissue culture supernatant fluids
are combined, resulting in a two-fold molar excess of hMN-14-Fab-DDD2.
The reaction mixture is incubated at room temperature for 24 hours under
mild reducing conditions using 1 mM reduced glutathione. Following
reduction, the DNL reaction is completed by mild oxidation using 2 mM
oxidized glutathione. The trimeric bispecific DNL construct is isolated
by affinity chromatography using an anti-idiotypic antibody against
hMN-14. The complex retains the binding affinities of both the hMN-14 and
hMN-15 antibodies.

[0370] Generation of Trimeric hPAM4-hMN-15 Bispecific DNL Construct

[0371] hMN-15-Fab-AD2 and hPAM4-Fab-DDD2 are prepared as described in
Example 11. The two fusion proteins (hPAM4-Fab-DDD2 and hMN-15-Fab-AD2)
are expressed independently in stably transfected myeloma cells. The
tissue culture supernatant fluids are combined, resulting in a two-fold
molar excess of hPAM4-Fab-DDD2. The reaction mixture is incubated at room
temperature for 24 hours under mild reducing conditions using 1 mM
reduced glutathione. Following reduction, the DNL reaction is completed
by mild oxidation using 2 mM oxidized glutathione. The trimeric
bispecific DNL construct is isolated by affinity chromatography using an
anti-idiotypic antibody against hMN-15. The complex retains the binding
affinities of both the hPAM4 and hMN-15 antibodies.

[0372] The skilled artisan will realize that a trimeric Fab construct can
be prepared using virtually any antibodies that have been cloned in an
expression vector, using appropriate restriction endonucleases and
standard molecular cloning techniques. Thus, combinations of hMN-15 with
any other antibody or antibody fragment may be prepared by DNL.

Example 18

Production of Targeting Peptides for Use in Pretargeting and 18F
Labeling

[0373] In certain embodiments, 18F-labeled proteins or peptides are
prepared by a novel technique and used for diagnostic and/or imaging
studies, such as PET imaging. The novel technique for 18F labeling
involves preparation of an 18F-metal complex, preferably an
18F-aluminum complex, which is chelated to a chelating moiety, such
as DOTA, NOTA or NETA or derivatives thereof. Chelating moieties may be
attached to proteins, peptides or any other molecule using conjugation
techniques well known in the art. In certain preferred embodiments, the
18F--Al complex is formed in solution first and then attached to a
chelating moiety that is already conjugated to a protein or peptide.
However, in alternative embodiments the aluminum may be first attached to
the chelating moiety and the 18F added later.

[0374] Peptide Synthesis

[0375] Peptides were synthesized by solid phase peptide synthesis using
the Fmoc strategy. Groups were added to the side chains of diamino amino
acids by using Fmoc/Aloc protecting groups to allow differential
deprotection. The Aloc groups were removed by the method of Dangles et.
al. (J. Org. Chem. 1987, 52:4984-4993) except that piperidine was added
in a 1:1 ratio to the acetic acid used. The unsymmetrical tetra-t-butyl
DTPA was made as described in McBride et al. (U.S. Pat. No. 7,405,320,
the Examples section of which is incorporated herein by reference).

[0384] F-18 Labeling of IMP 272--A 3 μL aliquot of the aluminum stock
solution was placed in a REACTI-VIALT® and mixed with 50 μL
18F (as received) and 3 μL of the IMP 272 solution. The solution
was heated in a heating block at 110° C. for 15 min and analyzed
by reverse phase HPLC. HPLC analysis (not shown) showed 93% free 18F
and 7% bound to the peptide. An additional 10 μL of the IMP 272
solution was added to the reaction and it was heated again and analyzed
by reverse phase HPLC (not shown). The HPLC trace showed 8% 18F at
the void volume and 92% of the activity attached to the peptide. The
remainder of the peptide solution was incubated at room temperature with
150 μL PBS for ˜1 hr and then examined by reverse phase HPLC.
The HPLC (not shown) showed 58% 18F unbound and 42% still attached
to the peptide. The data indicate that 18F--Al-DTPA complex may be
unstable when mixed with phosphate.

[0385] The labeled peptide was purified by applying the labeled peptide
solution onto a 1 cc (30 mg) WATERS® HLB column (Part #186001879) and
washing with 300 μL water to remove unbound F-18. The peptide was
eluted by washing the column with 2×100 μL 1:1 EtOH/H2O.
The purified peptide was incubated in water at 25° C. and analyzed
by reverse phase HPLC (not shown). The HPLC analysis showed that the
18F-labeled IMP 272 was not stable in water. After 40 min incubation
in water about 17% of the 18F was released from the peptide, while
83% was retained (not shown).

[0386] The peptide (16 μL 2 mM IMP 272, 48 μg) was labeled with
18F and analyzed for antibody binding by size exclusion HPLC. The
size exclusion HPLC showed that the peptide bound hMN-14×679 but
did not bind to the irrelevant bispecific antibody hMN-14×734 (not
shown).

[0387] IMP 27218F Labeling with Other Metals

[0388] A ˜3 μL aliquot of the metal stock solution (6×10
g mol) was placed in a polypropylene cone vial and mixed with 75
μL 18F (as received), incubated at room temperature for ˜2
min and then mixed with 20 μL of a 2 mM (4×10-8 mol) IMP
272 solution in 0.1 M NaOAc pH 4 buffer. The solution was heated in a
heating block at 100° C. for 15 min and analyzed by reverse phase
HPLC. IMP 272 was labeled with indium (24%), gallium (36%), zirconium
(15%), lutetium (37%) and yttrium (2%) (not shown). These results
demonstrate that the 18F metal labeling technique is not limited to
an aluminum ligand, but can also utilize other metals as well. With
different metal ligands, different chelating moieties may be utilized to
optimize binding of an F-18-metal conjugate.

[0389] Production and Use of a Serum-Stable 18F-Labeled Peptide IMP
449

[0390] The peptide, IMP 448 D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2
MH.sup.+ 1009 was made on Sieber Amide resin by adding the following
amino acids to the resin in the order shown: Aloc-D-Lys(Fmoc)-OH,
Trt-HSG-OH, the Aloc was cleaved, Fmoc-D-Tyr(But)-OH,
Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc was cleaved, Fmoc-D-Ala-OH with
final Fmoc cleavage to make the desired peptide. The peptide was then
cleaved from the resin and purified by HPLC to produce IMP 448, which was
then coupled to ITC-benzyl NOTA. The peptide, IMP 448, 0.0757 g
(7.5×10-5 mol) was mixed with 0.0509 g (9.09×10-5
mol) ITC benzyl NOTA and dissolved in 1 mL water. Potassium carbonate
anhydrous (0.2171 g) was then slowly added to the stirred peptide/NOTA
solution. The reaction solution was pH 10.6 after the addition of all the
carbonate. The reaction was allowed to stir at room temperature
overnight. The reaction was carefully quenched with 1 M HCl after 14 hr
and purified by HPLC to obtain 48 mg of IMP 449.

[0391]18F Labeling of IMP 449

[0392] The peptide IMP 449 (0.002 g, 1.37×10-6 mol) was
dissolved in 686 μL (2 mM peptide solution) 0.1 M NaOAc pH 4.02. Three
microliters of a 2 mM solution of Al in a pH 4 acetate buffer was mixed
with 15 μL, 1.3 mCi of 18F. The solution was then mixed with 20
μL of the 2 mM IMP 449 solution and heated at 105° C. for 15
min. Reverse phase HPLC analysis showed 35% (tR˜10 min) of the
activity was attached to the peptide and 65% of the activity was eluted
at the void volume of the column (3.1 min, not shown) indicating that the
majority of activity was not associated with the peptide. The crude
labeled mixture (5 μL) was mixed with pooled human serum and incubated
at 37° C. An aliquot was removed after 15 min and analyzed by
HPLC. The HPLC showed 9.8% of the activity was still attached to the
peptide (down from 35%). Another aliquot was removed after 1 hr and
analyzed by HPLC. The HPLC showed 7.6% of the activity was still attached
to the peptide (down from 35%), which was essentially the same as the 15
min trace (data not shown).

[0393] High Dose 18F Labeling

[0394] Further studies with purified IMP 449 demonstrated that the
18F-labeled peptide was highly stable (91%, not shown) in human
serum at 37° C. for at least one hour and was partially stable
(76%, not shown) in human serum at 37° C. for at least four hours.
Additional studies were performed in which the IMP 449 was prepared in
the presence of ascorbic acid as a stabilizing agent. In those studies
(not shown), the metal-18F-peptide complex showed no detectable
decomposition in serum after 4 hr at 37° C. The mouse urine 30 min
after injection of 18F-labeled peptide was found to contain 18F
bound to the peptide (not shown). These results demonstrate that the
18F-labeled peptides disclosed herein exhibit sufficient stability
under approximated in vivo conditions to be used for 18F imaging
studies.

[0395] For studies in the absence of ascorbic acid, 18F ˜21 mCi
in ˜400 μL of water was mixed with 9 μL of 2 mM AlCl3 in
0.1 M pH 4 NaOAc. The peptide, IMP 449, 60 μL (0.01 M,
6×10-7 mol in 0.5 NaOH pH 4.13) was added and the solution was
heated to 110° C. for 15 min. The crude labeled peptide was then
purified by placing the reaction solution in the barrel of a 1 cc
WATERS® HLB column and eluting with water to remove unbound 18F
followed by 1:1 EtOH/H20 to elute the 18F-labeled peptide. The
crude reaction solution was pulled through the column into a waste vial
and the column was washed with 3×1 mL fractions of water (18.97
mCi). The HLB column was then placed on a new vial and eluted with
2×200 μL 1:1 EtOH/H2O to collect the labeled peptide (1.83
mCi). The column retained 0.1 mCi of activity after all of the elutions
were complete. An aliquot of the purified 18F-labeled peptide (20
μL) was mixed with 200 μL of pooled human serum and heated at
37° C. Aliquots were analyzed by reverse phase HPLC. The results
showed the relative stability of 18F-labeled purified IMP 449 at
37° C. at time zero, one hour (91% labeled peptide), two hours
(77% labeled peptide) and four hours (76% labeled peptide) of incubation
in human serum (not shown). It was also observed that 18F-labeled
IMP 449 was stable in TFA solution, which is occasionally used during
reverse phase HPLC chromatography. There appears to be a general
correlation between stability in TFA and stability in human serum
observed for the exemplary 18F-labeled molecules described herein.
These results demonstrate that 18F-labeled peptide, produced
according to the methods disclosed herein, shows sufficient stability in
human serum to be successfully used for in vivo labeling and imaging
studies, for example using PET scanning to detect labeled cells or
tissues. Finally, since IMP 449 peptide contains a thiourea linkage,
which is sensitive to radiolysis, several products are observed by
RP-HPLC. However, when ascorbic acid is added to the reaction mixture,
the side products generated were markedly reduced.

Example 19

In Vivo Studies with Pretargeting Using MN-15-679 DNL Construct and
18F-Labeled Peptide

[0396]18F-labeled IMP 449 is prepared as follows. The 18F, 54.7
mCi in ˜0.5 mL is mixed with 3 μL 2 mM Al in 0.1 M NaOAc pH 4
buffer. After 3 min 10 μL of 0.05 M IMP 449 in 0.5 M pH 4 NaOAc buffer
is added and the reaction is heated in a 96° C. heating block for
15 min. The contents of the reaction are removed with a syringe. The
crude labeled peptide is then purified by HPLC on a C18 column. The
flow rate is 3 mL/min. Buffer A is 0.1% TFA in water and Buffer B is 90%
acetonitrile in water with 0.1% TFA. The gradient goes from 100% A to
75/25 A:B over 15 min. There is about 1 min difference in retention time
(tR) between the labeled peptide, which eluted first and the
unlabeled peptide. The HPLC eluent is collected in 0.5 min (mL)
fractions. The labeled peptide has a tR between 6 to 9 min depending
on the column used. The HPLC purified peptide sample is further processed
by diluting the fractions of interest two fold in water and placing the
solution in the barrel of a 1 cc WATERS® HLB column. The cartridge is
eluted with 3×1 mL water to remove acetonitrile and TFA followed by
400 μL 1:1 EtOH/H2O to elute the 18F-labeled peptide. The
purified [Al18F] IMP 449 elutes as a single peak on an analytical
HPLC C18 column (not shown).

[0397] Female athymic mice (nu/nu) bearing GW-39 xenografts are used for
in vivo studies. Three of the mice are injected with hMN-15-679 DNL
construct (162 μg) followed with [Al18F] IMP 449 18 h later. The
hMN-15-679 DNL construct is a humanized bispecific antibody of use for
tumor imaging studies, with divalent binding to the CEA tumor antigen and
monovalent binding to HSG. One mouse is injected with peptide alone. All
of the mice are necropsied at 1 h post peptide injection. Tissues are
counted immediately. Comparison of mean distributions shows substantially
higher levels of 18F-labeled peptide localized in the tumor than in
any normal tissues in the presence of tumor-targeting bispecific
antibody. The results demonstrate that 18F labeled peptide used in
conjunction with an hMN-15 containing antibody construct, such the
hMN-15-679 DNL construct, provide suitable targeting of the 18F
label to perform in vivo imaging, such as PET imaging analysis.

[0399] For example, Kinderman et al. (2006) examined the crystal structure
of the AD-DDD binding interaction and concluded that the human DDD
sequence contained a number of conserved amino acid residues that were
important in either dimer formation or AKAP binding, underlined in SEQ ID
NO:11 below. (See FIG. 1 of Kinderman et al., 2006.) The skilled artisan
will realize that in designing sequence variants of the DDD sequence, one
would desirably avoid changing any of the underlined residues, while
conservative amino acid substitutions might be made for residues that are
less critical for dimerization and AKAP binding.

[0400] Alto et al. (2003) performed a bioinformatic analysis of the AD
sequence of various AKAP proteins to design an RII selective AD sequence
called AKAP-IS (NO:13), with a binding constant for DDD of 0.4 nM. The
AKAP-IS sequence was designed as a peptide antagonist of AKAP binding to
PKA. Residues in the AKAP-IS sequence where substitutions tended to
decrease binding to DDD are underlined in SEQ ID NO:13 below.

TABLE-US-00012
AKAP-IS sequence
(SEQ ID NO: 13)
QIEYLAKQIVDNAIQQA

[0401] Similarly, Gold (2006) utilized crystallography and peptide
screening to develop a SuperAKAP-IS sequence (SEQ ID NO:27), exhibiting a
five order of magnitude higher selectivity for the RH isoform of PKA
compared with the R1 isoform. Underlined residues indicate the positions
of amino acid substitutions, relative to the AKAP-IS sequence, which
increased binding to the DDD moiety of RIM. In this sequence, the
N-terminal Q residue is numbered as residue number 4 and the C-terminal A
residue is residue number 20. Residues where substitutions could be made
to affect the affinity for RIM, were residues 8, 11, 15, 16, 18, 19 and
20 (Gold et al., 2006). It is contemplated that in certain alternative
embodiments, the SuperAKAP-IS sequence may be substituted for the AKAP-IS
AD moiety sequence to prepare cytokine-MAb DNL constructs. Other
alternative sequences that might be substituted for the AKAP-IS AD
sequence are shown in SEQ ID NO:28-30. Substitutions relative to the
AKAP-IS sequence are underlined. It is anticipated that, as with the
AKAP-IS sequence shown in SEQ ID NO:14, the AD moiety may also include
the additional N-terminal residues cysteine and glycine and C-terminal
residues glycine and cysteine.

[0403] Hundsrucker et al. (2006) developed still other peptide competitors
for AKAP binding to PKA, with a binding constant as low as 0.4 nM to the
DDD of the RII form of PKA. The sequences of various AKAP antagonistic
peptides is provided in Table 1 of Hundsrucker et al. (incorporated
herein by reference). Residues that were highly conserved among the AD
domains of different AKAP proteins are indicated below by underlining
with reference to the AKAP IS sequence (SEQ ID NO:13). The residues are
the same as observed by Alto et al. (2003), with the addition of the
C-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006),
incorporated herein by reference.) The sequences of peptide antagonists
with particularly high affinities for the RII DDD sequence are shown in
SEQ ID NO:34-36.

[0404] Carr et al. (2001) examined the degree of sequence homology between
different AKAP-binding DDD sequences from human and non-human proteins
and identified residues in the DDD sequences that appeared to be the most
highly conserved among different DDD moieties. These are indicated below
by underlining with reference to the human PKA RIIα DDD sequence of
SEQ ID NO:11. Residues that were particularly conserved are further
indicated by italics. The residues overlap with, but are not identical to
those suggested by Kinderman et al. (2006) to be important for binding to
AKAP proteins.

[0405] The skilled artisan will realize that in general, those amino acid
residues that are highly conserved in the DDD and AD sequences from
different proteins are ones that it may be preferred to remain constant
in making amino acid substitutions, while residues that are less highly
conserved may be more easily varied to produce sequence variants of the
AD and/or DDD sequences described herein.

[0406] The skilled artisan will realize that these and other amino acid
substitutions in the antibody moiety or linker portions of the DNL
constructs may be utilized to enhance the therapeutic and/or
pharmacokinetic properties of the resulting DNL constructs.

Example 21

Reactivity of MN-3 and MN-15 with CEA, NCA-90 and NCA-95 by Indirect Flow
Cytometry

[0407] The reacitivty of antibodies was tested by flow cytometry with a
Becton-Dickinson FACSCAN®. The biotinylated second antibody was
directed against mouse IgG and was detected by a
streptavidin/phycoerythrin conjugate. Tests were carried out with
antibodies at 1/50 dilution of 1 mg/ml stocks.

[0408] The antibodies MN-3 and MN-15 were tested for recognition of HeLa
cells transfected with cDNA coding for CEA (HeLa-CEA2), NCA-90
(HeLa-KNC6/S44) and NCA-95 (HeLa-CGM6/1). As a negative control, HeLa
cells transfected with the plasmid pSV2-Neo (control HeLa cells,
HeLa-NeoA) were used. In addition to the antibodies MN-3 and MN-15, three
control antibodies were included:

[0409] MAb 47 which cross reacts with CEA and NCA-95, but not NCA-90

[0410] MAb A which reacts with CEA

[0411] MAb NA which reacts with NCA-90

[0412] The results of the analysis by indirect fluorescence flow cytometry
showed that MN-3 binds NCA-90 and also CEA, but not NCA-95 (not shown).
In contrast, MN-15 binds to all three antigens--CEA, NCA-90 and NCA-95
(not shown). Control antibodies included in the panel tested showed the
expected cross-reactivities, as predicted from the literature (Berling et
al., Cancer Res 1990, 50:6534-39).